P-coumaroyl-CoA:monolignol transferases

ABSTRACT

The invention is directed to p-coumaroyl-CoA:monolignol transferase enzymes, nucleic acids encoding p-coumaroyl-CoA:monolignol transferase enzymes, and inhibitory nucleic acids adapted to inhibit the expression and/or translation of p-coumaroyl-CoA:monolignol transferase RNA; expression cassettes, plant cells, and plants that have or encode such nucleic acids and enzymes; and methods of making and using such nucleic acids, enzymes, expression cassettes, cells, and plants.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under DE-FC02-07ER64494awarded by the US Department of Energy. The government has certainrights in the invention.

FIELD OF THE INVENTION

The invention is directed to p-coumaroyl-CoA:monolignol transferaseenzymes, nucleic acids encoding p-coumaroyl-CoA:monolignol transferaseenzymes, and inhibitory nucleic acids adapted to inhibit the expressionand/or translation of p-coumaroyl-CoA:monolignol transferase RNA;expression cassettes, plant cells, and plants that have or encode suchnucleic acids and enzymes; and methods of making and using such nucleicacids, enzymes, expression cassettes, cells, and plants.

BACKGROUND

Lignin is an important cell wall component that provides structuralsupport to plants and is needed for plant vascular tissue function.Lignin is also a source of organic material for the synthesis ofchemicals. Lignin is one of the most abundant organic polymers on Earth,constituting about 30% of non-fossil organic carbon and from a quarterto a third of the dry mass of wood. Because the chemical structure oflignin is difficult to degrade by chemical and enzymatic means, ligninmakes the task of producing paper and biofuels from plant cell wallsdifficult. Modifying lignin to make it more amenable to degradation orsuitable for the production of certain chemicals is desirable.

SUMMARY OF THE INVENTION

The invention relates to the identification and isolation of newacyltransferase nucleic acids and polypeptides. The acyltransferases arep-coumaroyl-CoA:monolignol transferases (also called PMTs, or monolignolcoumarate transferases) that produce monolignol coumarates. Thep-coumaroyl-CoA:monolignol transferases can be used for making plantsthat contain modified lignin. The modified lignin is amenable todegradation and production of commodity chemicals.

One aspect of the invention is an isolated nucleic acid encoding ap-coumaroyl-CoA:monolignol transferase polypeptide with a SEQ ID NO:2 orSEQ ID NO:4 sequence.

Such p-coumaroyl-CoA:monolignol transferases can catalyze the synthesisof monolignol coumarate(s) from monolignol(s) and coumaroyl-CoA. Forexample, the monolignol can be coniferyl alcohol, p-coumaryl alcohol,sinapyl alcohol, or a combination thereof, and thep-coumaroyl-CoA:monolignol transferase can, for example, synthesizeconiferyl coumarate, p-coumaryl coumarate, sinapyl coumarate or acombination thereof. p-Coumaroyl-CoA:monolignol transferases with SEQ IDNO:2 or SEQ ID NO:4 sequences are unique in that they are selective forgenerating monolignol coumarates and have no relevant activity ingenerating monolignol ferulates. This is important as these transferasescan be used to generate modified lignin containing a higher proportionof monolignol coumarates without conducting other extraneous activity.

In some embodiments, the p-coumaroyl-CoA:monolignol transferase nucleicacid encodes a p-coumaroyl-CoA:monolignol transferase polypeptide with aSEQ ID NO:2 or SEQ ID NO:4 sequence. In other embodiments, the nucleicacids can, for example, encode a p-coumaroyl-CoA:monolignol transferasethat can catalyze the synthesis of monolignol coumarate(s) from amonolignol(s) and coumaroyl-CoA with at least about 50%, of the activityof a p-coumaroyl-CoA:monolignol transferase with the SEQ ID NO:2 or SEQID NO:4 amino acid sequence.

Another aspect of the invention is a transgenic plant cell comprising anisolated nucleic acid encoding a p-coumaroyl-CoA:monolignol transferase.The nucleic acid can include any of the p-coumaroyl-CoA:monolignoltransferase nucleic acids described herein. For example, the nucleicacid can include a nucleic acid segment that can selectively hybridizeto a DNA with a SEQ ID NO:1 or SEQ ID NO:3 sequence, or a nucleic acidthat encodes a SEQ ID NO:2 or SEQ ID NO:4 amino acid sequence, or anucleic acid that encodes a p-coumaroyl-CoA:monolignol transferase thatcan catalyze the synthesis of monolignol coumarate(s) from amonolignol(s) and coumaroyl-CoA with at least about 50%, of the activityof a p-coumaroyl-CoA:monolignol transferase with the SEQ ID NO:2 or SEQID NO:4 amino acid sequence.

Another aspect of the invention is an expression cassette comprising oneof the p-coumaroyl-CoA:monolignol transferase nucleic acids describedherein that is operably linked to a promoter functional in a host cell.Such a nucleic acid can include a nucleic acid segment that canselectively hybridize to a DNA with a SEQ ID NO:1 or SEQ ID NO:3sequence, or a nucleic acid that encodes a SEQ ID NO:2 or SEQ ID NO:4amino acid sequence, or a nucleic acid that encodes ap-coumaroyl-CoA:monolignol transferase that can catalyze the synthesisof monolignol coumarate(s) from a monolignol(s) and coumaroyl-CoA withat least about 50%, of the activity of a p-coumaroyl-CoA:monolignoltransferase with the SEQ ID NO:2 or SEQ ID NO:4 amino acid sequence. Theexpression cassette can further comprise a selectable marker gene. Insome embodiments, the expression cassette further comprises plasmid DNA.For example, the expression cassette can be within an expression vector.Promoters that can be used within such expression cassettes includepromoters functional during plant development or growth.

Another aspect of the invention is a plant cell that includes anexpression cassette comprising one of the p-coumaroyl-CoA:monolignoltransferase nucleic acids described herein that is operably linked to apromoter functional in a host cell. Such a nucleic acid can include anucleic acid segment that can selectively hybridize to a DNA with a SEQID NO:1 or SEQ ID NO:3 sequence, or a nucleic acid that encodes a SEQ IDNO:2 or SEQ ID NO:4 amino acid sequence, or a nucleic acid that encodesa p-coumaroyl-CoA:monolignol transferase that can catalyze the synthesisof monolignol coumarate(s) from a monolignol(s) and coumaroyl-CoA withat least about 50%, of the activity of a p-coumaroyl-CoA:monolignoltransferase with the SEQ ID NO:2 or SEQ ID NO:4 amino acid sequence. Theplant cell can be a monocot cell. The plant cell can also be agymnosperm cell. For example, the plant cell can be a maize, grass orsoftwood cell. In some embodiments, the plant cell is a dicot cell. Forexample, the plant cell can be a hardwood cell.

Another aspect of the invention is a plant that includes an expressioncassette comprising one of the p-coumaroyl-CoA:monolignol transferasenucleic acids described herein that is operably linked to a promoterfunctional in a host cell. Such a plant can be a monocot. Such a nucleicacid can include a nucleic acid segment that can selectively hybridizeto a DNA with a SEQ ID NO:1 or SEQ ID NO:3 sequence, or a nucleic acidthat encodes a SEQ ID NO:2 or SEQ ID NO:4 amino acid sequence, or anucleic acid that encodes a p-coumaroyl-CoA:monolignol transferase thatcan catalyze the synthesis of monolignol coumarate(s) from amonolignol(s) and coumaroyl-CoA with at least about 50%, of the activityof a p-coumaroyl-CoA:monolignol transferase with the SEQ ID NO:2 or SEQID NO:4 amino acid sequence. The plant can also be a gymnosperm. Forexample, the plant can be a maize, grass or softwood plant. In someembodiments, the plant is a dicot plant. For example, the plant can be ahardwood plant.

Another aspect of the invention is a method for incorporating monolignolcoumarates into lignin of a plant that includes:

-   -   a) stably transforming plant cells with the expression cassette        comprising one of the p-coumaroyl-CoA:monolignol transferase        nucleic acids described herein to generate transformed plant        cells;    -   b) regenerating the transformed plant cells into at least one        transgenic plant, wherein p-coumaroyl-CoA:monolignol transferase        is expressed in at least one transgenic plant in an amount        sufficient to incorporate monolignol coumarates into the lignin        of the transgenic plant.        For example, such a nucleic acid can be a nucleic acid that can        selectively hybridize to a DNA with a SEQ ID NO:1 or SEQ ID NO:3        sequence, or a nucleic acid that encodes a SEQ ID NO:2 or SEQ ID        NO:4 amino acid sequence, or a nucleic acid that encodes a        p-coumaroyl-CoA:monolignol transferase that can catalyze the        synthesis of monolignol coumarate(s) from a monolignol(s) and        coumaroyl-CoA with at least about 50%, of the activity of a        p-coumaroyl-CoA:monolignol transferase with the SEQ ID NO:2 or        SEQ ID NO:4 amino acid sequence. The method can be used to        generate a transgenic plant that is fertile. The method can        further include recovering transgenic seeds from the transgenic        plant, wherein the transgenic seeds include the nucleic acid        encoding a p-coumaroyl-CoA:monolignol transferase. The plant        containing monolignol coumarates within its lignin can be a        monocot. The plant can also be a gymnosperm. For example, the        plant can be a maize, grass or softwood plant. In some        embodiments, the plant is a dicot plant. For example, the plant        can also be a hardwood plant. Such a method can further include        stably transforming the plant cell(s) or the plant with at least        one selectable marker gene. The selectable marker can be linked        or associated with the expression cassette.

In some embodiments, the lignin in the plant that has the nucleic acidencoding a p-coumaroyl-CoA:monolignol transferase can include at least1% (wt %) monolignol coumarate. In other embodiments, the lignin in theplant can include at least 5% (wt %) monolignol coumarate, or at least10% (wt %) monolignol coumarate, or at least 20% (wt %) monolignolcoumarate, or at least 30% (wt %) monolignol coumarate, or at least 40%(wt %) monolignol coumarate, or at least 50% (wt %) monolignolcoumarate, or at least 60% (wt %) monolignol coumarate, or at least 70%(wt %) monolignol coumarate, or at least 80% (wt %) monolignolcoumarate, or at least 90% (wt %) monolignol coumarate, or about 100%(wt %) monolignol coumarate. In further embodiments, the lignin in theplant includes about 1-50% monolignol coumarate, or about 2-55%monolignol coumarate.

In some embodiments, the lignin in the plant that has the nucleic acidencoding a p-coumaroyl-CoA:monolignol transferase can be derived fromabout or at least 1% (mole %) monolignol coumarate. In otherembodiments, the lignin in the plant can include about or at least 5%(mole %) monolignol coumarate, or about or at least 10% (mole %)monolignol coumarate, or about or at least 20% (wt %) monolignolcoumarate, or about or at least 30% (mole %) monolignol coumarate, orabout or at least 40% (mole %) monolignol coumarate, or about or atleast 50% (mole %) monolignol coumarate, or about or at least 60% (mole%) monolignol coumarate, or about or at least 70% (mole %) monolignolcoumarate, or about or at least 80% (mole %) monolignol coumarate, orabout or at least 90% (mole %) monolignol coumarate, or about 100% (mole%) monolignol coumarate.

The method for incorporating monolignol coumarates into lignin of aplant can also include breeding the fertile transgenic plant to yield aprogeny plant, where the progeny plant has an increase in the percentageof monolignol coumarates in the lignin of the progeny plant relative tothe corresponding untransformed plant.

Another aspect of the invention is a lignin isolated from the transgenicplant comprising any of the p-coumaroyl-CoA:monolignol transferasenucleic acids described herein. Another aspect of the invention is awoody material isolated from the transgenic plant comprising any of thep-coumaroyl-CoA:monolignol transferase nucleic acids described herein.The lignin or woody tissue can include any of the nucleic acidsdescribed herein that encode a p-coumaroyl-CoA:monolignol transferase.In other embodiments, the lignin or woody tissue can include any of thep-coumaroyl-CoA:monolignol transferase amino acid or polypeptidesequences described herein.

Another aspect of the invention is a method of making a product from atransgenic plant comprising: (a) providing a transgenic plant thatincludes one of the isolated nucleic acids described herein that encodesa p-coumaroyl-CoA:monolignol transferase; and (b) processing thetransgenic plant's tissues under conditions sufficient to digest thelignin; to thereby generate the product from the transgenic plant,wherein the transgenic plant's tissues comprise lignin having anincreased percent of monolignol coumarates relative to a correspondinguntransformed plant. Such a corresponding untransformed plant istypically a plant of the same species, strain and/or accession as thetransformed plant. The conditions sufficient to digest the lignin caninclude conditions sufficient to cleave ester bonds within monolignolcoumarate-containing lignin. In some embodiments, the conditionssufficient to digest the lignin include mildly alkaline conditions. Insome embodiments, the conditions sufficient to digest the lignin includecontacting the transgenic plant's tissues with ammonia for a time and atemperature sufficient to cleave ester bonds within monolignolcoumarate-containing lignin. In some embodiments, the conditionssufficient to digest the lignin include acidic conditions.

Another aspect of the invention is an isolated nucleic acid encoding ap-coumaroyl-CoA:monolignol transferase, wherein the nucleic acid canselectively hybridize to a DNA with a SEQ ID NO:1 or SEQ ID NO:3sequence. For example, the nucleic acid can selectively hybridize to aDNA with a SEQ ID NO:1 or SEQ ID NO:3 sequence under stringenthybridization conditions. In some embodiments, the stringenthybridization conditions comprise a wash in 0.1×SSC, 0.1% SDS at 65° C.Such an isolated nucleic acid can have at least about 79%, at leastabout 80%, at least about 90%, or at least 95% sequence identity withSEQ ID NO:1 or SEQ ID NO:3. In some embodiments, the isolated nucleicacid with the SEQ ID NO:1 or SEQ ID NO:3 sequence encodes ap-coumaroyl-CoA:monolignol transferase.

Other aspects of the invention include inhibitory nucleic acids adaptedto inhibit expression and/or translation of a p-coumaroyl-CoA:monolignoltransferase mRNA; expression cassettes, plant cells, and plantscomprising the inhibitory nucleic acids; methods pertaining to the useof the inhibitory nucleic acids; transgenic plants comprising aknockdown or knockout of the plant's endogenousp-coumaroyl-CoA:monolignol transferase; and other aspects as describedin the following statements of the invention and elsewhere herein. Theseaspects of the invention can be carried out by adapting the descriptionsprovided in U.S. Pub. No. 2016/0046955, which is attached hereto and isincorporated herein by reference, to the sequences described herein.

Therefore, the invention embraces p-coumaroyl-CoA:monolignol transferaseenzymes, nucleic acids encoding or inhibiting expression ofp-coumaroyl-CoA:monolignol transferase enzymes, as well as expressioncassettes, plant cells, and plants that have or encode such nucleicacids and enzymes, and methods of making and using such nucleic acids,polypeptides, expression cassettes, cells, and plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A1, 1A2, 1B1 and 1B2 illustrate structural models for some typesof lignin polymers. FIGS. 1A1 and 1A2 show examples of lignin structuresthat may be found in a softwood (spruce). FIGS. 1B1 and 1B2 showexamples of lignin structures that may be present in a hardwood(poplar). [Ralph, J., Brunow, G., and Boerjan, W. (2007) Lignins. In:Rose, F., and Osborne, K. (eds). Encyclopedia of Life Sciences, DOI:10.1002/9780470015902.a0020104, John Wiley & Sons, Ltd., Chichester,UK]. The softwood lignin is generally more branched and contains a lowerproportion of β-ether units. Note that each of these structuresrepresents only one of billions of possible isomers [Ralph, J.,Lundquist, K., Brunow, G., Lu, F., Kim, H., Schatz, P. F., Marita, J.M., Hatfield, R. D., Ralph, S. A., Christensen, J. H., and Boerjan, W.Lignins: natural polymers from oxidative coupling of4-hydroxyphenylpropanoids. (2004) Phytochem. Revs. 3(1), 29-60]. Thus,these structures are merely illustrative of some of the linkage typesthat may be present different lignins. An “S” within a ring indicates asyringyl unit while a “G” within a unit indicates a guaiacyl unit.

FIGS. 2A-2E show the structures of possible reactants and products ofthe activity of certain PMT enzymes. FIG. 2A shows the structure ofsinapyl alcohol as a possible reactant. Coniferyl alcohol, anotherpossible reactant, lacks one of the two methoxy groups present onsinapyl alcohol. p-Hydroxycinnamyl alcohol (p-coumaryl alcohol), anotherpossible reactant, lacks both of the two methoxy groups present onsinapyl alcohol. FIG. 2B shows the structure of p-coumaroyl-CoA, anotherpossible reactant. FIG. 2C shows the structure of feruloyl-CoA, anotherpossible reactant. FIG. 2D shows the structure of sinapyl p-coumarate asa possible product resulting from the conjugation of sinapyl alcoholwith p-coumaryl-CoA. Coniferyl p-coumarate, a possible product resultingfrom the conjugation of coniferyl alcohol with p-coumaryl-CoA, lacks oneof the two methoxy groups present on sinapyl p-coumarate.p-Hydroxycinnamyl coumarate (p-coumaryl coumarate), a possible productresulting from the conjugation of p-hydroxycinnamyl alcohol andp-coumaryl-CoA, lacks both of the two methoxy groups present on sinapylp-coumarate. FIG. 2E shows the structure of sinapyl ferulate as apossible product resulting from the conjugation of sinapyl alcohol withferuloyl-CoA. Coniferyl ferulate, a possible product resulting from theconjugation of coniferyl alcohol with feruloyl-CoA, lacks one of the twomethoxy groups present on sinapyl ferulate. p-Hydroxycinnamyl ferulate(p-coumaryl ferulate), a possible product resulting from the conjugationof p-hydroxycinnamyl alcohol and feruloyl-CoA, lacks both of the twomethoxy groups present on sinapyl ferulate.

FIGS. 3A and 3B show liquid chromatography-mass spectrometry (LC-MS)traces of chemical species present after incubating a Panicum virgatum(switchgrass) PMT (PvPMT) with each of p-hydroxycinnamyl alcohol (H),coniferyl alcohol (G), and sinapyl alcohol (S) and either p-coumaryl-CoA(pCA-CoA) (FIG. 3A) or feruloyl-CoA (FA-CoA) (FIG. 3B).

FIGS. 4A and 4B show LC-MS traces of chemical species present afterincubating a Sorghum bicolor (sorghum) PMT (SbPMT) with each ofp-hydroxycinnamyl alcohol (H), coniferyl alcohol (G), and sinapylalcohol (S) and either p-coumaryl-CoA (pCA-CoA) (FIG. 4A) orferuloyl-CoA (FA-CoA) (FIG. 4B).

FIG. 5 shows LC-MS traces of chemical species present after incubating aZea mays (maize) PMT (ZmPMT) with each of p-hydroxycinnamyl alcohol (H),coniferyl alcohol (G), and sinapyl alcohol (S) and p-coumaryl-CoA(pCA-CoA).

FIG. 6 shows LC-MS traces of chemical species present after incubatingan Oryza sativa PMT (OsPMT) with each of p-hydroxycinnamyl alcohol (H),coniferyl alcohol (G), and sinapyl alcohol (S) and p-coumaryl-CoA(pCA-CoA).

FIG. 7 shows LC-MS traces of chemical species present after incubating aBrachypodium distachyon PMT (BdPMT1) with each of p-hydroxycinnamylalcohol (H), coniferyl alcohol (G), and sinapyl alcohol (S) andp-coumaryl-CoA (pCA-CoA).

FIG. 8 shows LC-MS traces of p-hydroxycinnamyl alcohol (H), coniferylalcohol (G), and sinapyl alcohol (S).

FIG. 9 shows LC-MS traces of chemical species present after incubatingeach of BdPMT, OsPMT, ZmPMT, PvPMT, and SbPMT with p-hydroxycinnamylalcohol (H), coniferyl alcohol (G), sinapyl alcohol (S), andp-coumaroyl-CoA. Products include sinapyl coumarate (S-pCA) andconiferyl coumarate (G-pCA).

FIG. 10 shows LC-MS traces of chemical species present after incubatingeach of OsFMT, BdPMT, OsPMT, ZmPMT, PvPMT, and SbPMT withp-hydroxycinnamyl alcohol (H), coniferyl alcohol (G), sinapyl alcohol(S), and feruloyl-CoA. Products include sinapyl ferulate (S-FA) andconiferyl ferulate (G-FA).

FIG. 11 is a schema showing a variety of compounds that can be made fromp-coumarate.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting. The following ispresented by way of illustration and does not limit the scope of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides nucleic acids and methods useful for alteringlignin structure and/or the lignin content in plants. Plants with suchaltered lignin structure/content are more easily or economicallyprocessed into useful products such as biofuels, paper, or commoditychemicals.

Acyl-CoA Dependent Acyltransferases

Plant acyl-CoA dependent acyltransferases constitute a large butspecific protein superfamily, named BAHD. Members of this family take anactivated carboxylic acid (i.e., a CoA thioester form of the acid) as anacyl donor and either an alcohol or, more rarely, a primary amine, as anacyl acceptor and catalyze the formation of an ester or an amide bond,respectively. The acyl donors and acyl acceptors that act as substratesby BAHD acyltransferases are quite diverse, and different BAHD familymembers exhibit a range of substrate specificities.

The invention relates to new BAHD acyltransferase nucleic acids andenzymes that enable the production of transgenic plants with alteredlignin. The BAHD nucleic acids can be used in the expression cassettes,expression vectors, transgenic plant cells, transgenic plants, andtransgenic seeds as described herein. The BAHD nucleic acids and encodedproteins are isolated or heterologous nucleic acids or proteins. Theterm “isolated” when used in conjunction with a nucleic acid orpolypeptide, refers to a nucleic acid segment or polypeptide that ispresent in a form or setting that is different from that in which it isfound in nature. For example, an isolated nucleic acid or an isolatedpolypeptide is identified and separated from at least one contaminantnucleic acid or polypeptide with which it is ordinarily associated inits natural state. In contrast, native nucleic acids, such as DNA, RNAand polypeptides are found in the state they exist in nature. The term“heterologous” when used in reference to a nucleic acid refers to anucleic acid segment that has been manipulated in some way. For example,a heterologous nucleic acid includes a nucleic acid segment from onespecies that has been introduced into another species. A heterologousnucleic acid also includes a nucleic acid segment that is native to anorganism that has been altered in some way (e.g., mutated, multiplecopies are added, the heterologous nucleic acid is linked to anon-native promoter or enhancer sequence, etc.). A heterologous nucleicacid also includes a nucleic acid comprising a combination of geneticelements not occurring in nature. Non-limiting examples of such geneticelements include coding sequences, promoters, enhancers, ribosomebinding sites (e.g., Shine Dalgarno sequences, Kozak sequences), etc.The term “heterologous” can also refer to any such individual geneticelement when included in such a non-naturally occurring combination.Heterologous nucleic acids can include plant nucleic acid segments suchas cDNA forms of a plant gene where the cDNA sequences are expressed ina sense direction to produce mRNA. In some embodiments, heterologousnucleic acids can be distinguished from endogenous plant genes in thatthe heterologous nucleic acid segments are joined to nucleotidesequences comprising regulatory elements such as promoters that are notfound naturally associated with the endogenous gene in its naturalchromosome. In some embodiments, heterologous nucleic acid can bedistinguished from endogenous plant genes in that the heterologousnucleic acid segments express the encoded protein (or portion of aprotein) in parts of the plant where the protein (or portion thereof) isnot normally expressed. The term “cDNA” refers to any DNA that includesa coding sequence for a polypeptide and lacks one or more intronspresent in naturally occurring genomic DNA also comprising that codingsequence, regardless of whether or not the cDNA is directly generatedfrom mRNA.

Feruloyl-CoA:monolignol transferases constitute one type of BAHDacyltransferases. These acyltransferases synthesize monolignol ferulatesfrom any of three monolignols (p-coumaryl, coniferyl, and sinapylalcohols). For example, feruloyl-CoA:monolignol transferases cansynthesize coniferyl ferulate from coniferyl alcohol and feruloyl-CoA,as shown below.

The feruloyl-CoA:monolignol transferases enable production of plantswith lignin that is readily cleaved and/or removed, for example, becausethe lignin in these plants contains monolignol ferulates such asconiferyl ferulate (CAFA). See Karlen, S. D.; Zhang, C.; Peck, M. L.;Smith, R. A.; Padmakshan, D.; Helmich, K. E.; Free, H. C. A.; Lee, S.;Smith, B. G.; Lu, F.; Sedbrook, J. C.; Sibout, R.; Grabber, J. H.;Runge, T. M.; Mysore, K. S.; Harris, P. J.; Bartley, L. E.; Ralph, J.,Monolignol ferulate conjugates are naturally incorporated into plantlignins. Science Advances 2016, 2 (10), e1600393.

The terms “feruloyl-CoA:monolignol transferase(s)” and “monolignolferulate transferase(s)” are used interchangeably herein.

Exemplary feruloyl-CoA:monolignol transferases are described in U.S.Appl. 62/481,281, U.S. Pat. Nos. 9,441,235, 9,487,794, 9,493,783, U.S.Pub. 2015/0020234A1, U.S. Pub. 2015/0307892A1, WO 2012/012698A1, WO2012/012741A1, and WO 2013/052660A1.

p-Coumaroyl-CoA:monolignol transferases (PMT, also called monolignolcoumarate transferases) constitute another type of BAHDacyltransferases. These acyltransferases catalyze the acylation ofmonolignols (e.g., p-coumaryl alcohol, coniferyl alcohol and/or sinapylalcohol) with p-coumarate, for example, as illustrated below.

Nucleic acids encoding p-coumaroyl-CoA:monolignol transferases of theinvention include nucleic acids encoding a Panicum virgatum(switchgrass) p-coumaroyl-CoA:monolignol transferase (PvPMT). Anexemplary nucleic acid encoding PvPMT has the following nucleic acidsequence (SEQ ID NO:1).

ATGGGTACCATCGGGTTCCCGGTGACGAGGACGAGCAGGTCGCTGGTGGCGCCGTCGTCGGCGACGCCGCAGGAGACGCTGCACCTGTCGGTGATCGACCGCGTGGCGGGGCTGCGGCACCTGGTGCGGTCGCTGCACGTGTTCGACGGCCGCCGCGGCGAGGCGGCGGTGAGGACGCCGGCGGAGACGCTGCGGGCGGCGCTGGGGAAGGCGCTGGTGGACTATTACCCGCTGGCGGGGCGGTTCGTGGAGGAGGACGGGGAGGTGCGGGTGGCGTGCACGGCGGGGGGCGCCTGGTTCGTGGAGGCGGCGGCGGCGTGCACCCTGGAGGAGGTGAAGCACCTGGACCACCCCATGGTCATCCCCAAGGAGGACCTGCTGCCGGAGCCGGCGCCGGACGTCAACCCCCTCGACATGCCGCTCATGATGCAGGTGACGGAGTTCGCGTGCGGCGGCTTCGTGGTGGGCCTCATCTCCGTGCACACCATCGCCGACGGCCTGGGCGCCGGGCAGTTCATCAACGCGGTGGCGGACTACGCGCGTGGCCTCCCGAGGCCCCGCGTGCTCCCCGTCTGGGCGCGCGACGTCATCCCGGCGCCGTCCAGGATCGTGTCCCCGCCGCCGCGGTTCGACCTCCTGGACCTCCGCTACTTCACCGTGGACCTCAGCCCGGAGCACATCGCCAAGGTCAAGTCCAGCTTCTTCGAGGCGACGGGGCAGCGCTGCTCGGCGTTCGACGTGTGCGTCGCCAAGACCTGGCAGTCCCGCGTCCGCGCGCTCCGGCTGGACGGCGACGACCCGGCGCGGCCCATCCACGTGTGCTTCTTCGCCAACACGCGGCACCTCCTGCCGCAGCTGGCGCCCGGGTTCTACGGCAACTGCTTCTACACCGTGAGGGCGACGCGGCCCTGCGGCGAGGTGGCGGCGGCCGGCGTGGTGGAGGTGGTGCGCGCCATCCGGGACGCCAAGGCGCGGCTGGGCGCGGACTTCGCGCGGTGGGCGGCGGGCGGGTTCGAGCGCGACCCCTACGAGCTCACCTTCAGCTACGACTCGCTCTTCGTCTCCGACTGGACGCGGCTGGGGTTCCTGGAGGCGGACTACGGGTGGGGCGCGCCGGCGCACGTCGTGCCCTTCTCCTACCACCCCTTCATGGCCGTCGCCGTCATCGGCGCGCCGCCGGCGCCCAAGCCCGGCGCGCGCGTCATGACCATGTGCGTCACGGAGAAGCACCTGCCCGAGTTCCAGGAGCAGATGAACGCCTTCGCCGCCGGGAA CCACCAGTGA

SEQ ID NO:1 encodes the following PvPMT amino acid sequence (SEQ IDNO:2).

MGTIGFPVTRTSRSLVAPSSATPQETLHLSVIDRVAGLRHLVRSLHVFDGRRGEAAVRTPAETLRAALGKALVDYYPLAGRFVEEDGEVRVACTAGGAWFVEAAAACTLEEVKHLDHPMVIPKEDLLPEPAPDVNPLDMPLMMQVTEFACGGFVVGLISVHTIADGLGAGQFINAVADYARGLPRPRVLPVWARDVIPAPSRIVSPPPRFDLLDLRYFTVDLSPEHIAKVKSSFFEATGQRCSAFDVCVAKTWQSRVRALRLDGDDPARPIHVCFFANTRHLLPQLAPGFYGNCFYTVRATRPCGEVAAAGVVEVVRAIRDAKARLGADFARWAAGGFERDPYELTFSYDSLFVSDWTRLGFLEADYGWGAPAHVVPFSYHPFMAVAVIGAPPAPKPGARVMTMCVTEKHLPEFQEQMNAFAAGNHQ*

Other nucleic acids encoding p-coumaroyl-CoA:monolignol transferases ofthe invention include nucleic acids encoding a Sorghum bicolor (sorghum)p-coumaroyl-CoA:monolignol transferase (SbPMT). An exemplary nucleicacid encoding SbPMT has the following nucleic acid sequence (SEQ IDNO:3).

ATGGGCACAATCGATGATACCGCCGGGTTATTCCCGGTGACGAGGACGAACAGGTCGCTGGTGCCGCCGTCGTCGGCGACGCCGCAGGAGACGCTGCGCCTGTCGGTGATCGACCGCGTGGCGGGGCTGCGCCACCTGGTGCGGTCGCTGCACGTGTTCGCCGGCGGCGAGAACAAGAAGCAGGCGGCGCCGCCGGCGAAGTCCCTGCGGGAGGCGCTGGGAAAGGCGCTGGTGGACTACTACCCGTTCGCGGGGCGGTTCGTGGAGGAAGACGGGGAGGTCCGGGTGGCGTGCACCGGCGAGGGCGCCTGGTTCGTGGAGGCCGCCGCCGCGTGCTCCCTGGAGGAGGTCCGGCACCTGGACCACCCCATGCTCATCCCCAAGGAGGAGCTGCTGCCGGAGCCGGCGCCCGGCGTCAACCCGCTCGACATGCCGCTCATGATGCAGGTGACGGAGTTCACGTGCGGCGGCTTCGTGGTGGGTCTAATCTCCGTCCACACCATCGCCGACGGTCTAGGCGCCGGGCAGTTCATCAACGCGGTGGCGGACTACGCCCGTGGCGGCGCCACCGCCGGCGCCGTCACCAGACCCCGCATCACCCCGATCTGGGCGCGCGACGTGATCCCGGACCCGCCCAAGATGCCGGCGCCGCCGCCGCGCCTCGACCTGCTGGACCTGGTCTACTTCACGACGGACCTGAGCCCGGACCACATCGCCAAGGTCAAGTCCAGCTACCTCGAGTCCACGGGGCAGCGCTGCTCGGCGTTCGACGTGTGCGTGGCGCGCACCTGGCAGGCCCGCGTCCGCGCGCTCCGCCTCCCGGACGCCGCCGCGCCCGTCCACGTCTGCTTCTTCGCCAACACCCGCCACCTGCTCCCGGCGACGGCGGCGGCGCCGGCGAGTGGGTTCTACGGCAACTGCTTCTACACCGTCAAGGCGACGCGGCCCAGCGGCGAGGTGGCGGCGGCCGACATCGTCGACGTCGTGCGCGCCATCCGGGACGCCAAGGCGAGGCTCGCCGCCGACTTCGCGAGGTGGGCGGCGGGCGGGTTTGATCGGGACCCCTACGAGCTCACCTTCACCTACGACTCCCTCTTCGTCTCCGACTGGACGAGGCTAGGGTTCCTCGAGGCTGACTATGGCTGGGGCACGCCGACGCACGTCGTGCCGTTCTCGTACCACCCGTTCATGGCCGTCGCCGTCATCGGGGCGCCGCCGGCGCCTAAGCCCGGCGCACGCATCATGACCATGTGCGTCCAGGAGCAGCACCTGCCTGAGTTCCAGGAGCAGATGAACCAGCCCTGCTCATGA

SEQ ID NO:3 encodes the following SbPMT amino acid sequence (SEQ IDNO:4).

MGTIDDTAGLFPVTRTNRSLVPPSSATPQETLRLSVIDRVAGLRHLVRSLHVFAGGENKKQAAPPAKSLREALGKALVDYYPFAGRFVEEDGEVRVACTGEGAWFVEAAAACSLEEVRHLDHPMLIPKEELLPEPAPGVNPLDMPLMMQVTEFTCGGFVVGLISVHTIADGLGAGQFINAVADYARGGATAGAVTRPRITPIWARDVIPDPPKMPAPPPRLDLLDLVYFTTDLSPDHIAKVKSSYLESTGQRCSAFDVCVARTWQARVRALRLPDAAAPVHVCFFANTRHLLPATAAAPASGFYGNCFYTVKATRPSGEVAAADIVDVVRAIRDAKARLAADFARWAAGGFDRDPYELTFTYDSLFVSDWTRLGFLEADYGWGTPTHVVPFSYHPFMAVAVIGAPPAPKPGARIMTMCVQEQHLPEFQEQMNQPCS*

The terms “p-coumaroyl-CoA:monolignol transferase(s)” and “monolignolcoumarate transferase(s)” are used interchangeably herein.

Nucleic acids encoding the aforementioned BAHD acyltransferases allowidentification and isolation of related nucleic acids and their encodedenzymes that provide a means for production of altered lignins inplants.

For example, related nucleic acids can be isolated and identified bymutation of the SEQ ID NO:1 or SEQ ID NO:3 sequence and/or byhybridization to DNA and/or RNA isolated from other plant species usingSEQ ID NO:1 or SEQ ID NO:3 nucleic acids as probes. The sequence of thep-coumaroyl-CoA:monolignol transferase enzyme (e.g., SEQ ID NO:2 or SEQID NO:4) can also be examined and used a basis for designing alternativep-coumaroyl-CoA:monolignol transferase nucleic acids that encode relatedp-coumaroyl-CoA:monolignol transferase polypeptides.

In one embodiment, the BAHD acyltransferase nucleic acids of theinvention include any nucleic acid that can selectively hybridize to SEQID NO:1 or SEQ ID NO:3.

The term “selectively hybridize” includes hybridization, under stringenthybridization conditions, of a nucleic acid sequence to a specifiednucleic acid target sequence (e.g., SEQ ID NO:1 or SEQ ID NO:3) to adetectably greater degree (e.g., at least 2-fold over background) thanits hybridization to non-target nucleic acid sequences. Such selectivehybridization substantially excludes non-target nucleic acids.Selectively hybridizing sequences typically have about at least 40%sequence identity, or at least 50% sequence identity, or at least 60%sequence identity, or at least 70% sequence identity, or 60-99% sequenceidentity, or 70-99% sequence identity, or 80-99% sequence identity, or90-95% sequence identity, or 90-99% sequence identity, or 95-97%sequence identity, or 97-99% sequence identity, or 100% sequenceidentity (or complementarity) with each other. In some embodiments, aselectively hybridizing sequence has at least about 70% or at leastabout 80% sequence identity or complementarity with SEQ ID NO:1 or SEQID NO:3.

Thus, the nucleic acids of the invention include those with about 500 ofthe same nucleotides as SEQ ID NO:1 or SEQ ID NO:3, or about 600 of thesame nucleotides as SEQ ID NO:1 or SEQ ID NO:3, or about 700 of the samenucleotides as SEQ ID NO:1 or SEQ ID NO:3, or about 800 of the samenucleotides as SEQ ID NO:1 or SEQ ID NO:3, or about 900 of the samenucleotides as SEQ ID NO:1 or SEQ ID NO:3, or about 1000 of the samenucleotides as SEQ ID NO:1 or SEQ ID NO:3, or about 1100 of the samenucleotides as SEQ ID NO: 1 or SEQ ID NO:3, or about 1200 of the samenucleotides as SEQ ID NO:1 or SEQ ID NO:3, or about 1300 of the samenucleotides as SEQ ID NO:1 or SEQ ID NO:3, or about 500-1325 of the samenucleotides as SEQ ID NO:1 or SEQ ID NO:3. The identical nucleotides oramino acids can be distributed throughout the nucleic acid or theprotein, and need not be contiguous.

Note that if a value of a variable that is necessarily an integer, e.g.,the number of nucleotides or amino acids in a nucleic acid or protein,is described as a range, e.g., 90-99% sequence identity what is meant isthat the value can be any integer between 90 and 99 inclusive, i.e., 90,91, 92, 93, 94, 95, 96, 97, 98 or 99, or any range between 90 and 99inclusive, e.g., 91-99%, 91-98%, 92-99%, etc.

The terms “stringent conditions” or “stringent hybridization conditions”include conditions under which a probe will hybridize to its targetsequence to a detectably greater degree than other sequences (e.g., atleast 2-fold over background). Stringent conditions are somewhatsequence-dependent and can vary in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified with up to 100%complementarity to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of sequence similarity are detected(heterologous probing). The probe can be approximately 20-500nucleotides in length, but can vary greatly in length from about 18nucleotides to equal to the entire length of the target sequence. Insome embodiments, the probe is about 10-50 nucleotides in length, orabout 18-25 nucleotides in length, or about 18-50 nucleotides in length,or about 18-100 nucleotides in length.

Typically, stringent conditions will be those where the saltconcentration is less than about 1.5 M Na ion (or other salts),typically about 0.01 to 1.0 M Na ion concentration (or other salts), atpH 7.0 to 8.3 and the temperature is at least about 30° C. for shorterprobes (e.g., 10 to 50 nucleotides) and at least about 60° C. for longerprobes (e.g., greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide or Denhardt's solution. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1MNaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1×SSC to2×SSC (where 20×SSC is 3.0 M NaCl, 0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40to 45% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in 0.5×SSC to1×SSC at 55 to 60° C. Exemplary high stringency conditions includehybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in0.1×SSC at 60 to 65° C. Specificity is typically a function ofpost-hybridization washes, where the factors controlling hybridizationinclude the ionic strength and temperature of the final wash solution.

For DNA-DNA hybrids, the T_(m) can be approximated from the equation ofMeinkoth and Wahl (Anal. Biochem. 138:267-84 (1984)):T _(m)=81.5° C.+16.6(log M)+0.41(% GC)−0.61(% formamide)−500/Lwhere M is the molarity of monovalent cations; % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % formamide is thepercentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. The T_(m) is reducedby about 1° C. for each 1% of mismatching. Thus, the T_(m),hybridization and/or wash conditions can be adjusted to hybridize tosequences of the desired sequence identity. For example, if sequenceswith greater than or equal to 90% sequence identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can include hybridizationand/or a wash at 1, 2, 3 or 4° C. lower than the thermal melting point(T_(m)). Moderately stringent conditions can include hybridizationand/or a wash at 6, 7, 8, 9 or 10° C. lower than the thermal meltingpoint (T_(m)). Low stringency conditions can include hybridizationand/or a wash at 11, 12, 13, 14, 15 or 20° C. lower than the thermalmelting point (T_(m)). Using the equation, hybridization and washcompositions, and a desired T_(m), those of ordinary skill can identifyand isolate nucleic acids with sequences related to SEQ ID NO:1 or SEQID NO:3.

Those of skill in the art also understand how to vary the hybridizationand/or wash solutions to isolate desirable nucleic acids. For example,if the desired degree of mismatching results in a T_(m) of less than 45°C. (aqueous solution) or 32° C. (formamide solution) it is preferred toincrease the SSC concentration so that a higher temperature can be used.

An extensive guide to the hybridization of nucleic acids is found inTijssen, LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY—HYBRIDIZATION WITH NUCLEIC ACID PROBES, part 1, chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” Elsevier, N.Y. (1993); and in CURRENT PROTOCOLS INMOLECULAR BIOLOGY, chapter 2, Ausubel, et al., eds, Greene Publishingand Wiley-Interscience, New York (1995).

Unless otherwise stated, in the present application high stringency isdefined as hybridization in 4×SSC, 5×Denhardt's (5 g Ficoll, 5 gpolyvinylpyrrolidone, 5 g bovine serum albumin in 500 mL of water), 0.1mg/mL boiled salmon sperm DNA, and 25 mM Na phosphate at 65° C., and awash in 0.1×SSC, 0.1% SDS at 65° C.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or nucleic acids or polypeptides: (a)“reference sequence,” (b) “comparison window,” (c) “sequence identity,”(d) “percentage of sequence identity” and (e) “substantial identity.”

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. The reference sequence can be a nucleicacid sequence (e.g., SEQ ID NO:1 or SEQ ID NO:3) or an amino acidsequence (e.g., SEQ ID NO:2 or SEQ ID NO:4). A reference sequence may bea subset or the entirety of a specified sequence. For example, areference sequence may be a segment of a full-length cDNA or of agenomic DNA sequence, or the complete cDNA or complete genomic DNAsequence, or a domain of a polypeptide sequence.

As used herein, “comparison window” refers to a contiguous and specifiedsegment of a nucleic acid or an amino acid sequence, wherein the nucleicacid/amino acid sequence can be compared to a reference sequence andwherein the portion of the nucleic acid/amino acid sequence in thecomparison window may comprise additions or deletions (i.e., gaps)compared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The comparisonwindow can vary for nucleic acid and polypeptide sequences. Generally,for nucleic acids, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100 or morenucleotides. For amino acid sequences, the comparison window is at leastabout 10 amino acids, and can optionally be 15, 20, 30, 40, 50, 100 ormore amino acids. Those of skill in the art understand that to avoid ahigh similarity to a reference sequence due to inclusion of gaps in thenucleic acid or amino acid sequence, a gap penalty is typicallyintroduced and is subtracted from the number of matches.

Methods of alignment of nucleotide and amino acid sequences forcomparison are well known in the art. The local homology algorithm(BESTFIT) of Smith and Waterman, (1981) Adv. Appl. Math 2:482, maypermit optimal alignment of compared sequences; by the homologyalignment algorithm (GAP) of Needleman and Wunsch, (1970) J. Mol. Biol.48:443-53; by the search for similarity method (Tfasta and Fasta) ofPearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444; bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, BLAST, FASTA and TFASTA in the WisconsinGenetics Software Package, Version 8 (available from Genetics ComputerGroup (GCG™ programs (Accelrys, Inc., San Diego, Calif.)). The CLUSTALprogram is well described by Higgins and Sharp (1988) Gene 73:237-44;Higgins and Sharp, (1989) CABIOS 5:151-3; Corpet, et al., (1988) NucleicAcids Res. 16:10881-90; Huang, et al., (1992) Computer Applications inthe Biosciences 8:155-65 and Pearson, et al., (1994) Meth. Mol. Biol.24:307-31. An example of a good program to use for optimal globalalignment of multiple sequences is PileUp (Feng and Doolittle, (1987) J.Mol. Evol., 25:351-60, which is similar to the method described byHiggins and Sharp, (1989) CABIOS 5:151-53 (and is hereby incorporated byreference). The BLAST family of programs that can be used for databasesimilarity searches includes: BLASTN for nucleotide query sequencesagainst nucleotide database sequences; BLASTX for nucleotide querysequences against protein database sequences; BLASTP for protein querysequences against protein database sequences; TBLASTN for protein querysequences against nucleotide database sequences; and TBLASTX fornucleotide query sequences against nucleotide database sequences. See,Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al.,eds., Greene Publishing and Wiley-Interscience, New York (1995). Anupdated version of the BLAST family of programs includes the BLAST+suite. (Camacho, C., Coulouris, G., Avagyan, V., Ma, N, Papadopoulos J,Bealer K, Madden T L. BLAST+: architecture and applications. BMCBioinformatics. 2009 Dec. 15; 10:421).

GAP uses the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443-53, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP makes a profit of gapcreation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package are 8 and 2, respectively. The gap creation and gapextension penalties can be expressed as an integer selected from thegroup of integers consisting of from 0 to 100. Thus, for example, thegap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40, 50 or more.

GAP presents one member of the family of best alignments. There may bemany members of this family. GAP displays four figures of merit foralignments: Quality, Ratio, Identity and Similarity. The Quality is themetric maximized in order to align the sequences. Ratio is the qualitydivided by the number of bases in the shorter segment. Percent Identityis the percent of the symbols that actually match. Percent Similarity isthe percent of the symbols that are similar. Symbols that are acrossfrom gaps are ignored. A similarity is scored when the scoring matrixvalue for a pair of symbols is greater than or equal to 0.50, thesimilarity threshold. The scoring matrix used in Version 10 of theWisconsin Genetics Software Package is BLOSUM62 (see, Henikoff andHenikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).

Sequence identity/similarity values provided herein can refer to thevalue obtained using the BLAST+2.5.0 suite of programs using defaultsettings (blast.ncbi.nlm.nih.gov) (Camacho, C., Coulouris, G., Avagyan,V., Ma, N, Papadopoulos J, Bealer K, Madden T L. BLAST+: architectureand applications. BMC Bioinformatics. 2009 Dec. 15; 10:421).

As those of ordinary skill in the art will understand, BLAST searchesassume that proteins can be modeled as random sequences. However, manyreal proteins comprise regions of nonrandom sequences, which may behomopolymeric tracts, short-period repeats, or regions enriched in oneor more amino acids. Such low-complexity regions may be aligned betweenunrelated proteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs can be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, (1993) Comput. Chem. 17:149-63) and XNU (C₁-ayerie andStates, (1993) Comput. Chem. 17:191-201) low-complexity filters can beemployed alone or in combination.

The terms “substantial identity” indicates that a polypeptide or nucleicacid comprises a sequence with between 55-100% sequence identity to areference sequence, with at least 55% sequence identity, or at least60%, or at least 70%, or at least 80%, or at least 90% or at least 95%sequence identity or any percentage of value within the range of 55-100%sequence identity relative to the reference sequence over a specifiedcomparison window. Optimal alignment may be ascertained or conductedusing the homology alignment algorithm of Needleman and Wunsch, supra.

An indication that two polypeptide sequences are substantially identicalis that both polypeptides have p-coumaroyl-CoA:monolignol transferaseactivity, meaning that both polypeptides can synthesize monolignolcoumarates from a monolignol and coumaroyl-CoA. The polypeptide that issubstantially identical to a p-coumaroyl-CoA:monolignol transferase witha SEQ ID NO:2 or SEQ ID NO:4 sequence may not have exactly the samelevel of activity as the p-coumaroyl-CoA:monolignol transferase with aSEQ ID NO:2 or SEQ ID NO:4. Instead, the substantially identicalpolypeptide may exhibit greater or lesser levels ofp-coumaroyl-CoA:monolignol transferase activity than thep-coumaroyl-CoA:monolignol transferase with SEQ ID NO:2 or SEQ ID NO:4,as measured by assays available in the art or described herein. Forexample, the substantially identical polypeptide can have at least about40%, or at least about 50%, or at least about 60%, or at least about70%, or at least about 80%, or at least about 90%, or at least about95%, or at least about 97%, or at least about 98%, or at least about100%, or at least about 105%, or at least about 110%, or at least about120%, or at least about 130%, or at least about 140%, or at least about150%, or at least about 200% of the activity of thep-coumaroyl-CoA:monolignol transferase with the SEQ ID NO:2 or SEQ IDNO:4 sequence when measured by similar assay procedures.

Alternatively, substantial identity is present when second polypeptideis immunologically reactive with antibodies raised against the firstpolypeptide (e.g., a polypeptide with SEQ ID NO:2 or SEQ ID NO:4). Thus,a polypeptide is substantially identical to a first polypeptide, forexample, where the two polypeptides differ only by a conservativesubstitution. In addition, a polypeptide can be substantially identicalto a first polypeptide when they differ by a non-conservative change ifthe epitope that the antibody recognizes is substantially identical.Polypeptides that are “substantially similar” share sequences as notedabove except that some residue positions, which are not identical, maydiffer by conservative amino acid changes.

The p-coumaroyl-CoA:monolignol transferase polypeptides of the presentinvention may include the first 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99 N-terminalamino acid residues of the SEQ ID NO:2 or SEQ ID NO:4 sequence.Alternatively, the p-coumaroyl-CoA:monolignol transferase polypeptidesof the present invention may include the first 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98and 99 C-terminal amino acid residues of the SEQ ID NO:2 or SEQ ID NO:4sequence.

Lignin

Lignin broadly refers to a biopolymer that is typically part ofsecondary cell walls in plants. Lignin is a complex moderatelycross-linked aromatic polymer (see, e.g., FIG. 1). Lignin may also becovalently linked to hemicelluloses. Hemicellulose broadly refers to aclass of branched sugar polymers composed of pentoses and hexoses.Hemicelluloses typically have an amorphous structure with up to hundredsor thousands of pentose units and they are generally at least partiallysoluble in dilute alkali. Cellulose broadly refers to an organiccompound with the formula (C₆H₁₀O₅)_(z) where z is an integer. Celluloseis a linear polysaccharide that can include linear chains ofbeta-1-4-linked glucose residues of several hundred to over ten thousandunits.

Lignocellulosic biomass represents an abundant, inexpensive, and locallyavailable feedstock for conversion to carbonaceous fuel (e.g., ethanol,biodiesel, biofuel and the like). However, the complex structure oflignin, which includes ether and carbon-carbon bonds that bind togetherthe various subunits of lignin, and the crosslinking of lignin to otherplant cell wall polymers, make it the most recalcitrant of plantpolymers. Thus, significant quantities of lignin in a biomass caninhibit the efficient usage of plants as a source of fuels and othercommercial products. Gaining access to the carbohydrate andpolysaccharide polymers of plant cells for use as carbon and energysources therefore requires significant energy input and often harshchemical treatments, especially when significant amounts of lignin arepresent. For example, papermaking procedures in which lignin is removedfrom plant fibers by delignification reactions are typically expensive,can be polluting and generally require use of high temperatures andharsh chemicals largely because the structure of lignin is impervious tomild conditions. Plants with altered lignin structures that could bemore readily cleaved under milder conditions would reduce the costs ofpapermaking and make the production of biofuels more competitive withcurrently existing procedures for producing oil and gas fuels.

Plants make lignin from a variety of subunits or monomers that aregenerally termed monolignols. Such primary monolignols includep-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol.

Monolignols destined for lignin polymerization in normal plants can bepreacylated with acetate, p-hydroxybenzoate, or p-coumarate (Ralph etal., Phytochem. Rev. 3:29-60 (2004)). p-Coumarates can acylate theγ-position of phenylpropanoid side chains mainly found in the syringylunits of lignin. Studies indicate that monolignols, primarily sinapylalcohol, are enzymatically preacylated with p-coumarate prior to theirincorporation into lignin, indicating that the monolignol p-coumarateconjugates, coniferyl p-coumarate and sinapyl p-coumarate, can also be‘monomer’ precursors of lignin.

Although monolignol p-coumarate-derived units may comprise up to 40% ofthe lignin in some grass tissues, the p-coumarate moiety from suchconjugates does not enter into the radical coupling (polymerization)reactions occurring during lignification. Instead, the p-coumaratemoieties substantially remain as terminal units with an unsaturated sidechain and a free phenolic group (Ralph et al., J. Am. Chem. Soc. 116:9448-9456 (1994); Hatfield et al., J. Sci. Food Agric. 79: 891-899(1999)). Thus, the presence of sinapyl p-coumarate conjugates produces alignin ‘core’ with terminal p-coumarate groups and no new bonds in thebackbone of the lignin polymer.

Regardless, lignocellulosic biomass with lignin comprising a higherproportion of p-coumarate content is more amenable to pretreatment andsaccharification (hydrolysis). Pretreatment of biomass removes a largeproportion of the lignin and other materials from the cellulose andhemicellulose and enhances the porosity of the biomass for optionaldownstream hydrolysis. A variety of biomass pretreatments are well knownin the art. Exemplary pretreatments include chipping, grinding, milling,steam pretreatment, ammonia fiber expansion (AFEX, also referred to asammonia fiber explosion), ammonia recycle percolation (ARP), CO₂explosion, steam explosion, ozonolysis, wet oxidation, acid hydrolysis,dilute-acid hydrolysis, alkaline hydrolysis, organosolv, extractiveammonia (EA) pretreatment, and pulsed electrical field treatment, amongothers. See, e.g., Kumar et al. 2009 (Kumar, P.; Barrett, D. M.;Delwiche, M. J.; Stroeve, P., Methods for Pretreatment ofLignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production.Industrial & Engineering Chemistry Research 2009, 48, (8), 3713-3729)and da Costa Sousa et al. 2016 (da Costa Sousa, L.; Leonardo, Jin, M.;Chundawat, S. P. S.; Bokade, V.; Tang, X.; Azarpira, A.; Lu, F.; Avci,F.; Humpula, J.; Uppugundla, N.; Gunawan, C.; Pattathil, S.; Cheh, A.M.; Kothari, N.; Kumar, N.; Ralph, J.; Hahn, M. G.; Wyman, C. E.; Singh,S.; Simmons, B. A.; Dale, B. E.; Balan, V. Next-Generation AmmoniaPretreatment Enhances Cellulosic Biofuel Production. Energy Environ.Sci., 2016, 9, 1215-1223). Hydrolysis converts biomass polymers tofermentable sugars, such as glucose and xylose, and other monomeric oroligomeric components. Methods for hydrolyzing biomass, also known assaccharification, are well known in the art. Exemplary hydrolysismethods include enzymatic hydrolysis (e.g., with cellulases or otherenzymes) and acid hydrolysis (e.g., with sulfurous, sulfuric,hydrochloric, hydrofluoric, phosphoric, nitric, and/or formic acids),among other methods. Thus, plants and biomass with lignin comprising ahigher proportion of p-coumarate content are more suitable to processingfor downstream applications.

Lignin comprising a higher proportion of p-coumarate content also has ahigher proportion of pendant p-coumarate units, which can be cleavedfrom the lignin using conditions typically employed for cleaving esterbonds, described in further detail below. The cleaved p-coumarate unitscan be recovered for downstream uses.

p-Coumarate (or p-coumaric acid), currently valued at ˜$20/kg, has somesignificant applications, but, because has not been previously availablein bulk quantities, its applications have been limited. This couldreadily change with the p-coumarate-enriched lignin provided with thepresent invention. p-Coumarate has a number of medical/cosmetic uses.See, e.g., U.S. Pub. No. 2007/0183996 A1, U.S. Pub. No. 2007/0183996 A1,U.S. Pat. Nos. 8,481,593, 9,089,499, U.S. Pub. No. 2007/0183996, U.S.Pub. No. 2011/0237551, and U.S. Pub. No. 2013/0272983). p-Coumarate alsohas a large number of applications in health, food, pharmaceutical, andcosmetic industries due to its physiological functions in antioxidant,anti-mutagenesis, anti-genotoxicity, antimicrobial, anti-inflammatory,anti-melanogenesis, and anti-thrombosis activities. See Ferguson et al.2003 (Ferguson, L. R., Lim, I. F., Pearson, A. E., Ralph, J., andHarris, P. J. Bacterial antimutagenesis by hydroxycinnamic acids fromplant cell walls. (2003) Mutation Research-Genetic Toxicology andEnvironmental Mutagenesis 542(1-2), 49-58), Ferguson et al. 2005(Ferguson, L. R., Zhu, S. T., and Harris, P. J. Antioxidant andantigenotoxic effects of plant cell wall hydroxycinnamic acids incultured HT-29 cells. (2005) Molecular Nutrition & Food Research 49(6),585-593), Bodini et al. (Bodini, S. F., Manfredini, S., Epp, M.,Valentini, S., and Santori, F. Quorum sensing inhibition activity ofgarlic extract and p-coumaric acid. (2009) Lett Appl Microbiol 49(5),551-555), An et al. 2008 (An, S. M., Lee, S. I., Choi, S. W., Moon, S.W., and Boo, Y. C. p-Coumaric acid, a constituent of Sasa quelpaertensisNakai, inhibits cellular melanogenesis stimulated by alpha-melanocytestimulating hormone. (2008) Brit J Dermatol 159(2), 292-299), andRazzaghi-Asl et al. 2013 (Razzaghi-Asl, N., Garrido, J., Khazraei, H.,Borges, F., and Firuzi, O. Antioxidant properties of hydroxycinnamicacids: A review of structure-activity relationships. (2013) CurrentMedicinal Chemistry 20(36), 4436-4450). p-Coumarate is also used as aprecursor for natural aromatic organic compounds, includingp-hydroxybenzoic acid and 4-vinylphenol, or a variety of commoditychemicals, including caffeate (Nambudiri A M, Bhat J V. Conversion ofp-coumarate into caffeate by Streptomyces nigrifaciens. Purification andproperties of the hydroxylating enzyme. Biochem J. 1972 November;130(2):425-33), volatile phenols (Cabrita M J P V, Patao R, Freitas A MC. Conversion of hydroxycinnamic acids into volatile phenols in asynthetic medium and red wine by Dekkera bruxellensis. Ciencia eTecnologia de Alimentos, Campinas. 2012; 32(1):106-11), and others. FIG.11 shows a variety of derivatives that are readily produced fromp-coumarate.

p-Coumarate is also a versatile and attractive building block for thegeneration of novel, sustainable polymeric materials. The phenolicfunctional groups allow these building blocks to be used as monomers instep- and chain-polymerization reactions (Upton, B. M., and Kasko, A. M.Strategies for the conversion of lignin to high-value polymericmaterials: Review and perspective. (2016) Chemical Reviews 116(4),2275-2306). Derivatives have been used for the synthesis of polyesters,where they replace petroleum-based diols (Kaneko, T., Matsusaki, M.,Hang, T. T., and Akashi, M. Thermotropic liquid-crystalline polymerderived from natural cinnamoyl biomonomers. (2004) Macromol Rapid Comm25(5), 673-677) (Nagata, M., and Hizakae, S. Synthesis andcharacterization of photocrosslinkable biodegradable polymers derivedfrom 4-hydroxycinnamic acid. (2003) Macromol Biosci 3(8), 412-419).Thermal polymerization of p-coumaric acid, for example, affords aliquid-crystalline polymer that adopts a nematic liquid-crystallinestructure at temperatures between 215-280° C. (Kaneko, T., Matsusaki,M., Hang, T. T., and Akashi, M. Thermotropic liquid-crystalline polymerderived from natural cinnamoyl biomonomers. (2004) Macromol Rapid Comm25(5), 673-677). Methacrylation of certain lignin-derived monomers hasprovided access to monomers that can be polymerized using conventionalfree-radical polymerization methods as well as via various controlledradical polymerization techniques, including atom transfer radicalpolymerization (ATRP) and reversible addition fragmentation chaintransfer (RAFT) polymerization (Holmberg, A. L., Reno, K. H., Nguyen, N.A., Wool, R. P., and Epps, T. H., 3rd. Syringyl methacrylate, a hardwoodlignin-based monomer for high-Tg polymeric materials. (2016) ACS MacroLetters 5(5), 574-578).

In contrast to p-coumarate, ferulate esters do undergo radical couplingreactions under lignification conditions. Model ferulates, such as theferulate shown below (where R is CH₃—, CH₃—CH₂—, a sugar, apolysaccharide, pectin, cell-wall (arabino)xylan or other plantcomponent), readily undergo radical coupling reactions with each otherand with lignin monomers and oligomers to form cross-linked networks.

If present during lignification, ferulates can become inextricably boundinto the lignin by ether and C—C bonds. Although such ferulate moietiesare no more extractable or cleavable from the lignin structure thanother lignin units under most conditions, the ester itself can bereadily cleaved using conditions generally employed for ester cleavage.Upon cleavage of such ester bonds, other plant cell wall components canbe released. For example, an arabinoxylan (hemicellulose) chain can bereleased from a ferulate-mediated lignin attachment by cleaving theester.

Ferulate-monolignol ester conjugates, such as coniferyl ferulate orsinapyl ferulate, are made by plants as secondary metabolites during,among other things, lignin biosynthesis. [Paula et al, Tetrahedron 51:12453-12462 (1994); Seca et al., Phytochemistry 56: 759-767 (2001);Hsiao & Chiang, Phytochemistry 39: 899-902 (1995); Li et al., PlantaMed. 72: 278-280 (2005)]. The structures of coniferyl ferulate andsinapyl ferulate are shown below.

Feruloyl-CoA:monolignol transferases biosynthesize coniferyl ferulatefrom coniferyl alcohol and feruloyl-CoA as shown below.

The incorporation of monolignol ferulates into the lignin of plantsallows the cell wall materials and lignin to be readily cleaved orprocessed into useful products. See also, U.S. Pat. Nos. 9,441,235,9,487,794, and 9,493,783, the contents of all of which are specificallyincorporated herein by reference in their entireties.

Monolignol ferulates made by feruloyl-CoA:monolignol transferases can beincorporated by radical coupling into plant lignins. Both the monolignoland the ferulate moieties can undergo such coupling, resulting in alignin that can be complex. However, such ‘double-ended-incorporation’still yields readily cleavable ester linkages that have been engineeredinto the backbone of the lignin polymer network. Esters are readilycleaved under much less stringent conditions by the same chemicalprocesses used to cleave lignin, but the lignin resulting from themethods described herein is significantly easier to cleave, and providesmore facile and less costly access to the plant cell wallpolysaccharides. See also, U.S. Pat. Nos. 9,441,235, 9,487,794, and9,493,783, the contents of all of which are specifically incorporatedherein by reference in their entireties.

Lignins can be degraded by chemical or enzymatic means to yield avariety of smaller monomers and oligomers. While enzymatic processes aregenerally preferred because they do not require high temperatures andharsh chemicals, such enzymatic processes have previously not been aseffective at solubilizing lignin moieties away from valuable plant cellconstituents (e.g., polysaccharides and carbohydrates).

Plants with the feruloyl-CoA:monolignol transferase nucleic acids and/orenzymes supply monolignol ferulates for facile lignification in plants,thereby yielding plants with lignins that are more readily cleaved orprocessed to release cellulose, hemicelluloses and lignin breakdownproducts.

Conditions for releasing the cellulose, hemicelluloses and ligninbreakdown products from plants containing the feruloyl-CoA:monolignoltransferase nucleic acids and/or enzymes include conditions typicallyemployed for cleaving ester bonds. Thus, the ester bonds withinmonolignol ferulate-rich lignins can be cleaved by milder alkalineand/or acidic conditions than the conditions typically used to breakdown the lignin of plants that are not rich in monolignol ferulates. Forexample, mildly alkaline conditions involving use of ammonia may be usedto cleave the ester bonds within monolignol ferulate-rich lignins,whereas such conditions would not cleave substantially any of the etherand carbon-carbon bonds in normal lignins. See also, U.S. patentapplication Ser. No. 12/830,905, filed Jul. 6, 2010 and to U.S. PatentApplication Ser. No. 61/213,706, filed Jul. 6, 2009, the contents ofboth of which are specifically incorporated herein by reference in theirentireties.

For acid digestion, exemplary methods include but are not limited toacid γ-valerolactone acid digestion (Luterbacher, J. S., Azarpira, A.,Motagamwala, A. H., Lu, F., Ralph, J., and Dumesic, J. A. Aromaticmonomer production integrated into the γ-valerolactone sugar platform.(2015) Energy and Environmental Science 8(9), 2657-2663), digestion asdescribed in Santoro et al. (Santoro, N., Cantu, S. L., Tornqvist, C.E., Falbel, T. G., Bolivar, J. L., Patterson, S. E., Pauly, M., andWalton, J. D. A high-throughput platform for screening milligramquantities of plant biomass for lignocellulose digestibility. (2010)Bioenergy Research 3(1), 93-102), and ionic digestion (Kim, K. H.,Dutta, T., Ralph, J., Mansfield, S. D., Simmons, B. A., and Singh, S.Impact of lignin polymer backbone esters on ionic liquid pretreatment ofpoplar. (2017) Biotechnology for Biofuels).

Plants Modified to Contain a p-Coumaroyl-CoA:Monolignol Transferase

In order to engineer plants with lignins that contain significant levelsof monolignol coumarates, one of skill in the art can introducep-coumaroyl-CoA:monolignol transferases or nucleic acids encoding suchp-coumaroyl-CoA:monolignol transferases into the plants. For example,one of skill in the art can inject p-coumaroyl-CoA:monolignoltransferase enzymes into young plants.

Alternatively, one of skill in the art can generate genetically-modifiedplants that contain nucleic acids encoding p-coumaroyl-CoA:monolignoltransferases within their somatic and/or germ cells. Such geneticmodification can be accomplished by procedures available in the art. Forexample, one of skill in the art can prepare an expression cassette orexpression vector that can express one or more encodedp-coumaroyl-CoA:monolignol transferase enzymes. Plant cells can betransformed by the expression cassette or expression vector, and wholeplants (and their seeds) can be generated from the plant cells that weresuccessfully transformed with the p-coumaroyl-CoA:monolignol transferasenucleic acids. Some procedures for making such genetically modifiedplants and their seeds are described below.

Promoters:

The p-coumaroyl-CoA:monolignol transferase nucleic acids of theinvention can be operably linked to a promoter, which provides forexpression of mRNA from the p-coumaroyl-CoA:monolignol transferasenucleic acids. The promoter is typically a promoter functional in plantsand/or seeds, and can be a promoter functional during plant growth anddevelopment. A p-coumaroyl-CoA:monolignol transferase nucleic acid isoperably linked to the promoter when it is located downstream from thepromoter, to thereby form an expression cassette.

Most endogenous genes have regions of DNA that are known as promoters,which regulate gene expression. Promoter regions are typically found inthe flanking DNA upstream from the coding sequence in both prokaryoticand eukaryotic cells. A promoter sequence provides for regulation oftranscription of the downstream gene sequence and typically includesfrom about 50 to about 2,000 nucleotide base pairs. Promoter sequencesalso contain regulatory sequences such as enhancer sequences that caninfluence the level of gene expression. Some isolated promoter sequencescan provide for gene expression of heterologous DNAs, that is a DNAdifferent from the native or homologous DNA.

Promoter sequences are also known to be strong or weak, or inducible. Astrong promoter provides for a high level of gene expression, whereas aweak promoter provides for a very low level of gene expression. Aninducible promoter is a promoter that provides for the turning on andoff of gene expression in response to an exogenously added agent, or toan environmental or developmental stimulus. For example, a bacterialpromoter such as the P_(tac) promoter can be induced to vary levels ofgene expression depending on the level of isothiopropylgalactoside addedto the transformed cells. Promoters can also provide for tissue specificor developmental regulation. An isolated promoter sequence that is astrong promoter for heterologous DNAs is advantageous because itprovides for a sufficient level of gene expression for easy detectionand selection of transformed cells and provides for a high level of geneexpression when desired.

Suitable promoters for use in the present invention include native orheterologous promoters.

Expression cassettes generally include, but are not limited to, a plantpromoter such as the CaMV 35S promoter (Odell et al., Nature.313:810-812 (1985)), or others such as CaMV 19S (Lawton et al., PlantMolecular Biology. 9:315-324 (1987)), nos (Ebert et al., Proc. Natl.Acad. Sci. USA. 84:5745-5749 (1987)), Adhl (Walker et al., Proc. Natl.Acad. Sci. USA. 84:6624-6628 (1987)), sucrose synthase (Yang et al.,Proc. Natl. Acad. Sci. USA. 87:4144-4148 (1990)), α-tubulin, ubiquitin,actin (Wang et al., Mol. Cell. Biol. 12:3399 (1992)), cab (Sullivan etal., Mol. Gen. Genet. 215:431 (1989)), PEPCase (Hudspeth et al., PlantMolecular Biology. 12:579-589 (1989)) or those associated with the Rgene complex (Chandler et al., The Plant Cell. 1:1175-1183 (1989)).Further suitable promoters include the poplar xylem-specific secondarycell wall specific cellulose synthase 8 promoter, cauliflower mosaicvirus promoter, the Z10 promoter from a gene encoding a 10 kD zeinprotein, a Z27 promoter from a gene encoding a 27 kD zein protein,inducible promoters, such as the light inducible promoter derived fromthe pea rbcS gene (Coruzzi et al., EMBO J. 3:1671 (1971)) and the actinpromoter from rice (McElroy et al., The Plant Cell. 2:163-171 (1990)).Seed specific promoters, such as the phaseolin promoter from beans, mayalso be used (Sengupta-Gopalan, Proc. Natl. Acad. Sci. USA. 83:3320-3324(1985). Further suitable promoters include any of the promoters on thevarious genes of the conventional lignin monomer (monolignol)biosynthetic pathway. See, e.g., Vanholme et al. 2012 (Vanholme, R.,Morreel, K., Darrah, C., Oyarce, P., Grabber, J. H., Ralph, J., andBoerjan, W. Metabolic engineering of novel lignin in biomass crops.(2012) New Phytol. 196(4), 978-1000); Vanholme et al. 2010 (Vanholme,R., Demedts, B., Morreel, K., Ralph, J., and Boerjan, W. Ligninbiosynthesis and structure. (2010) Plant Physiol. 153(3), 895-905),Vanholme et al. 2008 (Vanholme, R., Morreel, K., Ralph, J., and Boerjan,W. Lignin engineering. (2008) Curr. Opin. Plant Biol. 11(3), 278-285),Boerjan et al. 2003 (Boerjan, W., Ralph, J., and Baucher, M. Ligninbiosynthesis. (2003) Annual Reviews in Plant Biology 54, 519-546). Anexemplary promoter from this pathway is the cinnamate-4-hydroxylase(C4H) promoter (Bell-Lelong, D. A., Cusumano, J. C., Meyer, K., andChapple, C. Cinnamate-4-hydroxylase expression in Arabidopsis:regulation in response to development and the environment. (1997) PlantPhysiol. 113, 729-738), the sequence of which is SEQ ID NO:5:

aagcttagaggagaaactgagaaaatcagcgtaatgagagacgagagcaatgtgctaagagaagagattgggaagagagaagagacgataaaggaaacggaaaagcatatggaggagcttcatatggagcaagtgaggctgagaagacggtcgagtgagcttacggaagaagtggaaaggacgagagtgtctgcatcggaaatggctgagcagaaaagagaagctataagacagctttgtatgtctcttgaccattacagagatgggtacgacaggctttggagagttgttgccggccataagagtaagagagtagtggttttaacaacttgaagtgtaagaacaatgagtcaatgactacgtgcaggacattggacataccgtgtgttcttttggattgaaatgttgtttcgaagggctgttagttgatgttgaaaataggttgaagttgaataatgcatgttgatatagtaaatatcaatggtaatattttctcatttcccaaaactcaaatgatatcatttaattataaactaacgtaaactgttgacaatacacttatggttaaaaatttggagtcttgttttagtatacgtatcaccaccgcacggtttcaaaaccacataattgtaaatgttattggaaaaaagaacccgcaatacgtattgtattttggtaaacatagctctaagcctctaatatataagctctcaacaattctggctaatggtcccaagtaagaaaagcccatgtattgtaaggtcatgatctcaaaaacgagggtgaggtggaatactaacatgaggagaaagtaaggtgacaaatttttggggcaatagtggtggatatggtggggaggtaggtagcatcatttctccaagtcgctgtctttcgtggtaatggtaggtgtgtctctctttatattatttattactactcattgttaatttctttttttctacaatttgtttcttactccaaaatacgtcacaaatataatactaggcaaataattatttaattgtaagtcaatagagtggttgttgtaaaattgatttttgatattgaaagagttcatggacggatgtgtatgcgccaaatgctaagcccttgtagtcttgtactgtgccgcgcgtatattttaaccaccactagttgtttctctttttcaaaaacacacaaaaaataatttgttttcgtaacggcgtcaaatctgacggcgtctcaatacgttcaattttttctttctttcacatggtttctcatagctttgcattgaccataggtaaagggataaggataaaggttttttctcttgtttgttttatccttattattcaaaatggataaaaaaacagtcttattttgatttctttgattaaaaaagtcattgaaattcatatttgattttttgctaaatgtcaactcagagacacaaacgtaatgcactgtcgccaatattcatggatcatgaccatgaatatcactagaataattgaaaatcagtaaaatgcaaacaaagcattttctaattaaaacagtcttctacattcacttaattggaatttcctttatcaaacccaaagtccaaaacaatcggcaatgttttgcaaaatgttcaaaactattggcgggttggtctatccgaattgaagatcttttctccatatgatagaccaacgaaattcggcatacgtgtttttttttttgttttgaaaaccctttaaacaaccttaattcaaaatactaatgtaactttattgaacgtgcatctaaaaattttgaactttgcttttgagaaataatcaatgtaccaataaagaagatgtagtacatacattataattaaatacaaaaaaggaatcaccatatagtacatggtagacaatgaaaaactttaaaacatatacaatcaataatactctttgtgcataactttttttgtcgtctcgagtttatatttgagtacttatacaaactattagattacaaactgtgctcagatacattaagttaatcttatatacaagagcactcgagtgttgtccttaagttaatcttaagatatcttgaggtaaatagaaatagttaactcgtttttattttcttttttttaccatgagcaaaaaaagatgaagtaagttcaaaacgtgacgaatctacatgttactacttagtatgtgtcaatcattaaatcgggaaaacttcatcatttcaggagtactacaaaactcctaagagtgagaacgactacatagtacatattttgataaaagacttgaaaacttgctaaaacgaatttgcgaaaatataatcatacaagtagaaccactgatttgatcgaattattcatagctttgtaggatgaacttaactaaataatatctcacaaaagtattgacagtaacctagtactatactatctatgttagaatatgattatgatataatttatcccctcacttattcatatgatttttgaagcaactactttcgtttttttaacattttcttttttggtttttgttaatgaacatatttagtcgtttcttaattccactcaaatagaaaatacaaagagaactttatttaatagatatgaacataatctcacatcctcctcctaccttcaccaaacacttttacatacactttgtggtctttctttacctaccaccatcaacaacaacaccaagccccactcacacacacgcaatcacgttaaatctaacgccgtttattatctcatcattcaccaactcccacgtacctaacgccgtttaccttttgccgttggtcctcatttctcaaaccaaccaaacctctccctcttataaaatcctctctcccttctttatttcttcctcagcagcttcttctgctttcaattactctcgccgacgattttctcaccggaaaaaaacaatatcattgcggata cacaaactataOther promoters useful in the practice of the invention are known tothose of skill in the art.

Alternatively, novel tissue specific promoter sequences may be employedin the practice of the present invention. cDNA clones from a particulartissue can be isolated and those clones which are expressed specificallyin that tissue are identified, for example, using Northern blotting.Preferably, the gene isolated not present in a high copy number, but isrelatively abundant in specific tissues. The promoter and controlelements of corresponding genomic clones can then be localized usingtechniques well known to those of skill in the art.

A p-coumaroyl-CoA:monolignol transferase nucleic acid can be combinedwith the promoter by standard methods to yield an expression cassette,for example, as described in Sambrook et al. (MOLECULAR CLONING: ALABORATORY MANUAL. Second Edition (Cold Spring Harbor, N.Y.: Cold SpringHarbor Press (1989); MOLECULAR CLONING: A LABORATORY MANUAL. ThirdEdition (Cold Spring Harbor, N.Y.: Cold Spring Harbor Press (2000)).Briefly, a plasmid containing a promoter such as the 35S CaMV promotercan be constructed as described in Jefferson (Plant Molecular BiologyReporter 5:387-405 (1987)) or obtained from Clontech Lab in Palo Alto,Calif. (e.g., pBI121 or pBI221). Typically, these plasmids areconstructed to have multiple cloning sites having specificity fordifferent restriction enzymes downstream from the promoter. Thep-coumaroyl-CoA:monolignol transferase nucleic acids can be subcloneddownstream from the promoter using restriction enzymes and positioned toensure that the DNA is inserted in proper orientation with respect tothe promoter so that the DNA can be expressed as sense RNA. Once thep-coumaroyl-CoA:monolignol transferase nucleic acid is operably linkedto a promoter, the expression cassette so formed can be subcloned into aplasmid or other vector (e.g., an expression vector).

In some embodiments, a cDNA clone encoding a p-coumaroyl-CoA:monolignoltransferase protein is isolated from a selected plant tissue, or anucleic acid encoding a mutant or modified p-coumaroyl-CoA:monolignoltransferase protein is prepared by available methods or as describedherein. For example, the nucleic acid encoding a mutant or modifiedp-coumaroyl-CoA:monolignol transferase protein can be any nucleic acidwith a coding region that hybridizes to SEQ ID NO:1 or SEQ ID NO:3 andthat has p-coumaroyl-CoA:monolignol transferase activity. Usingrestriction endonucleases, the entire coding sequence for thep-coumaroyl-CoA:monolignol transferase is subcloned downstream of thepromoter in a 5′ to 3′ sense orientation.

Targeting Sequences:

Additionally, expression cassettes can be constructed and employed totarget the p-coumaroyl-CoA:monolignol transferase nucleic acids to anintracellular compartment within plant cells or to direct an encodedprotein to the extracellular environment. This can generally be achievedby joining a DNA sequence encoding a transit or signal peptide sequenceto the coding sequence of the p-coumaroyl-CoA:monolignol transferasenucleic acid. The resultant transit, or signal, peptide will transportthe protein to a particular intracellular, or extracellular destination,respectively, and can then be posttranslational removed. Transitpeptides act by facilitating the transport of proteins throughintracellular membranes, e.g., vacuole, vesicle, plastid andmitochondrial membranes, whereas signal peptides direct proteins throughthe extracellular membrane. By facilitating transport of the proteininto compartments inside or outside the cell, these sequences canincrease the accumulation of a particular gene product in a particularlocation. For example, see U.S. Pat. No. 5,258,300.

3′ Sequences:

When the expression cassette is to be introduced into a plant cell, theexpression cassette can also optionally include 3′ nontranslated plantregulatory DNA sequences that act as a signal to terminate transcriptionand allow for the polyadenylation of the resultant mRNA. The 3′nontranslated regulatory DNA sequence preferably includes from about 300to 1,000 nucleotide base pairs and contains plant transcriptional andtranslational termination sequences. For example, 3′ elements that canbe used include those derived from the nopaline synthase gene ofAgrobacterium tumefaciens (Bevan et al., Nucleic Acid Research.11:369-385 (1983)), or the terminator sequences for the T7 transcriptfrom the octopine synthase gene of Agrobacterium tumefaciens, and/or the3′ end of the protease inhibitor I or II genes from potato or tomato.Other 3′ elements known to those of skill in the art can also beemployed. These 3′ nontranslated regulatory sequences can be obtained asdescribed in An (Methods in Enzymology. 153:292 (1987)). Many such 3′nontranslated regulatory sequences are already present in plasmidsavailable from commercial sources such as Clontech, Palo Alto, Calif.The 3′ nontranslated regulatory sequences can be operably linked to the3′ terminus of the p-coumaroyl-CoA:monolignol transferase nucleic acidsby standard methods.

Selectable and Screenable Marker Sequences:

In order to improve identification of transformants, a selectable orscreenable marker gene can be employed with the expressiblep-coumaroyl-CoA:monolignol transferase nucleic acids. “Marker genes” aregenes that impart a distinct phenotype to cells expressing the markergene and thus allow such transformed cells to be distinguished fromcells that do not have the marker. Such genes may encode either aselectable or screenable marker, depending on whether the marker confersa trait which one can ‘select’ for by chemical means, i.e., through theuse of a selective agent (e.g., a herbicide, antibiotic, or the like),or whether it is simply a trait that one can identify throughobservation or testing, i.e., by ‘screening’ (e.g., the R-locus trait).Of course, many examples of suitable marker genes are known to the artand can be employed in the practice of the invention.

Included within the terms selectable or screenable marker genes are alsogenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which encode a secretable antigen that can be identifiedby antibody interaction, or secretable enzymes that can be detected bytheir catalytic activity. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., byELISA; and proteins that are inserted or trapped in the cell wall (e.g.,proteins that include a leader sequence such as that found in theexpression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a polypeptide that becomes sequestered in the cell wall, wherethe polypeptide includes a unique epitope may be advantageous. Such asecreted antigen marker can employ an epitope sequence that wouldprovide low background in plant tissue, a promoter-leader sequence thatimparts efficient expression and targeting across the plasma membrane,and can produce protein that is bound in the cell wall and yet isaccessible to antibodies. A normally secreted wall protein modified toinclude a unique epitope would satisfy such requirements.

Example of proteins suitable for modification in this manner includeextensin or hydroxyproline rich glycoprotein (HPRG). For example, themaize HPRG (Stiefel et al., The Plant Cell. 2:785-793 (1990)) is wellcharacterized in terms of molecular biology, expression, and proteinstructure and therefore can readily be employed. However, any one of avariety of extensins and/or glycine-rich wall proteins (Keller et al.,EMBO J. 8:1309-1314 (1989)) could be modified by the addition of anantigenic site to create a screenable marker.

Numerous other possible selectable and/or screenable marker genes willbe apparent to those of skill in the art in addition to the one setforth herein below. Therefore, it will be understood that the discussionherein is exemplary rather than exhaustive. In light of the techniquesdisclosed herein and the general recombinant techniques that are knownin the art, the present invention readily allows the introduction of anygene, including marker genes, into a recipient cell to generate atransformed plant cell, e.g., a monocot cell or dicot cell.

Possible selectable markers for use in connection with the presentinvention include, but are not limited to, a neo gene (Potrykus et al.,Mol. Gen. Genet. 199:183-188 (1985)) which codes for kanamycinresistance and can be selected for using kanamycin, G418, and the like;a bar gene which codes for bialaphos resistance; a gene which encodes analtered EPSP synthase protein (Hinchee et al., Bio/Technology. 6:915-922(1988)) thus conferring glyphosate resistance; a nitrilase gene such asbxn from Klebsiella ozaenae which confers resistance to bromoxynil(Stalker et al., Science. 242:419-423 (1988)); a mutant acetolactatesynthase gene (ALS) which confers resistance to imidazolinone,sulfonylurea or other ALS-inhibiting chemicals (European PatentApplication 154,204 (1985)); a methotrexate-resistant DHFR gene (Thilletet al., J. Biol. Chem. 263:12500-12508 (1988)); a dalapon dehalogenasegene that confers resistance to the herbicide dalapon; or a mutatedanthranilate synthase gene that confers resistance to 5-methyltryptophan. Where a mutant EPSP synthase gene is employed, additionalbenefit may be realized through the incorporation of a suitablechloroplast transit peptide, CTP (European Patent Application 0 218 571(1987)).

An illustrative embodiment of a selectable marker gene capable of beingused in systems to select transformants is the gene that encode theenzyme phosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes (U.S. Pat. No. 5,550,318). The enzyme phosphinothricinacetyl transferase (PAT) inactivates the active ingredient in theherbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutaminesynthetase, (Murakami et al., Mol. Gen. Genet. 205:42-50 (1986); Twellet al., Plant Physiol. 91:1270-1274 (1989)) causing rapid accumulationof ammonia and cell death. The success in using this selective system inconjunction with monocots was surprising because of the majordifficulties that have been reported in transformation of cereals(Potrykus, Trends Biotech. 7:269-273 (1989)).

Screenable markers that may be employed include, but are not limited to,a J3-glucuronidase or uidA gene (GUS) that encodes an enzyme for whichvarious chromogenic substrates are known; an R-locus gene, which encodesa product that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta et al., In: Chromosome Structure andFunction: Impact of New Concepts, 18^(th) Stadler Genetics Symposium, J.P. Gustafson and R. Appels, eds. (New York: Plenum Press) pp. 263-282(1988)); a j3-lactamase gene (Sutcliffe, Proc. Natl. Acad. Sci. USA.75:3737-3741 (1978)), which encodes an enzyme for which variouschromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci.USA. 80:1101 (1983)) which encodes a catechol dioxygenase that canconvert chromogenic catechols; an α-amylase gene (Ikuta et al.,Bio/technology 8:241-242 (1990)); a tyrosinase gene (Katz et al., J.Gen. Microbiol. 129:2703-2714 (1983)) which encodes an enzyme capable ofoxidizing tyrosine to DOPA and dopaquinone which in turn condenses toform the easily detectable compound melanin; a β-galactosidase gene,which encodes an enzyme for which there are chromogenic substrates; aluciferase (lux) gene (Ow et al., Science. 234:856-859.1986), whichallows for bioluminescence detection; or an aequorin gene (Prasher etal., Biochem. Biophys. Res. Comm. 126:1259-1268 (1985)), which may beemployed in calcium-sensitive bioluminescence detection, or a green oryellow fluorescent protein gene (Niedz et al., Plant Cell Reports.14:403 (1995)).

For example, genes from the maize R gene complex can be used asscreenable markers. The R gene complex in maize encodes a protein thatacts to regulate the production of anthocyanin pigments in most seed andplant tissue. Maize strains can have one, or as many as four, R allelesthat combine to regulate pigmentation in a developmental and tissuespecific manner. A gene from the R gene complex does not harm thetransformed cells. Thus, an R gene introduced into such cells will causethe expression of a red pigment and, if stably incorporated, can bevisually scored as a red sector. If a maize line carries dominantalleles for genes encoding the enzymatic intermediates in theanthocyanin biosynthetic pathway (C2, A1, A2, Bz1 and Bz2), but carriesa recessive allele at the R locus, transformation of any cell from thatline with R will result in red pigment formation. Exemplary linesinclude Wisconsin 22 that contains the rg-Stadler allele and TR112, aK55 derivative that is r-g, b, Pl. Alternatively any genotype of maizecan be utilized if the C1 and R alleles are introduced together.

The R gene regulatory regions may be employed in chimeric constructs inorder to provide mechanisms for controlling the expression of chimericgenes. More diversity of phenotypic expression is known at the R locusthan at any other locus (Coe et al., in Corn and Corn Improvement, eds.Sprague, G. F. & Dudley, J. W. (Am. Soc. Agron., Madison, Wis.), pp.81-258 (1988)). It is contemplated that regulatory regions obtained fromregions 5′ to the structural R gene can be useful in directing theexpression of genes, e.g., insect resistance, drought resistance,herbicide tolerance or other protein coding regions. For the purposes ofthe present invention, it is believed that any of the various R genefamily members may be successfully employed (e.g., P, S, Lc, etc.).However, one that can be used is Sn (particularly Sn:bol3). Sn is adominant member of the R gene complex and is functionally similar to theR and B loci in that Sn controls the tissue specific deposition ofanthocyanin pigments in certain seedling and plant cells, therefore, itsphenotype is similar to R.

A further screenable marker contemplated for use in the presentinvention is firefly luciferase, encoded by the lux gene. The presenceof the lux gene in transformed cells may be detected using, for example,X-ray film, scintillation counting, fluorescent spectrophotometry,low-light video cameras, photon counting cameras or multiwellluminometry. It is also envisioned that this system may be developed forpopulation screening for bioluminescence, such as on tissue cultureplates, or even for whole plant screening.

Other Optional Sequences:

An expression cassette of the invention can also further compriseplasmid DNA. Plasmid vectors include additional DNA sequences thatprovide for easy selection, amplification, and transformation of theexpression cassette in prokaryotic and eukaryotic cells, e.g.,pUC-derived vectors such as pUC8, pUC9, pUC18, pUC19, pUC23, pUC119, andpUC120, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors,or pBS-derived vectors. The additional DNA sequences include origins ofreplication to provide for autonomous replication of the vector,additional selectable marker genes, preferably encoding antibiotic orherbicide resistance, unique multiple cloning sites providing formultiple sites to insert DNA sequences or genes encoded in theexpression cassette and sequences that enhance transformation ofprokaryotic and eukaryotic cells.

Another vector that is useful for expression in both plant andprokaryotic cells is the binary Ti plasmid (as disclosed in Schilperoortet al., U.S. Pat. No. 4,940,838) as exemplified by vector pGA582. Thisbinary Ti plasmid vector has been previously characterized by An(Methods in Enzymology. 153:292 (1987)) and is available from Dr. An.This binary Ti vector can be replicated in prokaryotic bacteria such asE. coli and Agrobacterium. The Agrobacterium plasmid vectors can be usedto transfer the expression cassette to dicot plant cells, and undercertain conditions to monocot cells, such as rice cells. The binary Tivectors preferably include the nopaline T DNA right and left borders toprovide for efficient plant cell transformation, a selectable markergene, unique multiple cloning sites in the T border regions, the colE1replication of origin and a wide host range replicon. The binary Tivectors carrying an expression cassette of the invention can be used totransform both prokaryotic and eukaryotic cells, but is preferably usedto transform dicot plant cells.

In Vitro Screening of Expression Cassettes:

Once the expression cassette is constructed and subcloned into asuitable plasmid, it can be screened for the ability to substantiallyinhibit the translation of an mRNA coding for a seed storage protein bystandard methods such as hybrid arrested translation. For example, forhybrid selection or arrested translation, a preselected antisense DNAsequence is subcloned into an SP6/T7 containing plasmids (as supplied byProMega Corp.). For transformation of plants cells, suitable vectorsinclude plasmids such as described herein. Typically, hybrid arresttranslation is an in vitro assay that measures the inhibition oftranslation of an mRNA encoding a particular seed storage protein. Thisscreening method can also be used to select and identify preselectedantisense DNA sequences that inhibit translation of a family orsubfamily of zein protein genes. As a control, the corresponding senseexpression cassette is introduced into plants and the phenotype assayed.

DNA Delivery of the DNA Molecules into Host Cells:

The present invention generally includes steps directed to introducingp-coumaroyl-CoA:monolignol transferase nucleic acids, such as apreselected cDNA encoding the selected p-coumaroyl-CoA:monolignoltransferase enzyme, into a recipient cell to create a transformed cell.In some instances the frequency of occurrence of cells taking upexogenous (foreign) DNA may be low. Moreover, it is most likely that notall recipient cells receiving DNA segments or sequences will result in atransformed cell wherein the DNA is stably integrated into the plantgenome and/or expressed. Some may show only initial and transient geneexpression. However, certain cells from virtually any dicot or monocotspecies may be stably transformed, and these cells regenerated intotransgenic plants, through the application of the techniques disclosedherein.

Another aspect of the invention is a plant with lignin containingmonolignol coumarates (e.g., coniferyl coumarate), wherein the plant hasan introduced p-coumaroyl-CoA:monolignol transferase nucleic acid. Theplant can be a monocotyledon or a dicotyledon. Another aspect of theinvention includes plant cells (e.g., embryonic cells or other celllines) that can regenerate fertile transgenic plants and/or seeds. Thecells can be derived from either monocotyledons or dicotyledons.Suitable examples of plant species include grasses (switchgrass,sorghum, etc.), softwoods, hardwoods, wheat, rice, Arabidopsis, tobacco,maize, soybean, sorghum, and the like. In some embodiments, the plant orcell is a monocotyledon plant or cell. For example, the plant or cellcan be a softwood plant or cell, or a maize plant or cell. In someembodiments, the plant or cell is a dicotyledon plant or cell. Forexample, the plant or cell can be a hardwood plant or cell. The cell(s)may be in a suspension cell culture or may be in an intact plant part,such as an immature embryo, or in a specialized plant tissue, such ascallus, such as Type I or Type II callus.

Transformation of the plant cells can be conducted by any one of anumber of methods known to those of skill in the art. Examples are:Transformation by direct DNA transfer into plant cells byelectroporation (U.S. Pat. Nos. 5,384,253 and 5,472,869, Dekeyser etal., The Plant Cell. 2:591-602 (1990)); direct DNA transfer to plantcells by PEG precipitation (Hayashimoto et al., Plant Physiol.93:857-863 (1990)); direct DNA transfer to plant cells bymicroprojectile bombardment (McCabe et al., Bio/Technology. 6:923-926(1988); Gordon-Kamm et al., The Plant Cell. 2:603-618 (1990); U.S. Pat.Nos. 5,489,520; 5,538,877; and 5,538,880) and DNA transfer to plantcells via infection with Agrobacterium. Methods such as microprojectilebombardment or electroporation can be carried out with “naked” DNA wherethe expression cassette may be simply carried on any E. coli-derivedplasmid cloning vector. In the case of viral vectors, it is desirablethat the system retain replication functions, but lack functions fordisease induction.

One method for dicot transformation, for example, involves infection ofplant cells with Agrobacterium tumefaciens using the leaf-disk protocol(Horsch et al., Science 227:1229-1231 (1985). Monocots such as Zea mayscan be transformed via microprojectile bombardment of embryogenic callustissue or immature embryos, or by electroporation following partialenzymatic degradation of the cell wall with a pectinase-containingenzyme (U.S. Pat. Nos. 5,384,253; and 5,472,869). For example,embryogenic cell lines derived from immature Zea mays embryos can betransformed by accelerated particle treatment as described byGordon-Kamm et al. (The Plant Cell. 2:603-618 (1990)) or U.S. Pat. Nos.5,489,520; 5,538,877 and 5,538,880, cited above. Excised immatureembryos can also be used as the target for transformation prior totissue culture induction, selection and regeneration as described inU.S. application Ser. No. 08/112,245 and PCT publication WO 95/06128.Furthermore, methods for transformation of monocotyledonous plantsutilizing Agrobacterium tumefaciens have been described by Hiei et al.(European Patent 0 604 662, 1994) and Saito et al. (European Patent 0672 752, 1995).

Methods such as microprojectile bombardment or electroporation arecarried out with “naked” DNA where the expression cassette may be simplycarried on any E. coli-derived plasmid cloning vector. In the case ofviral vectors, it is desirable that the system retain replicationfunctions, but lack functions for disease induction.

The choice of plant tissue source for transformation will depend on thenature of the host plant and the transformation protocol. Useful tissuesources include callus, suspensions, culture cells, protoplasts, leafsegments, stem segments, tassels, pollen, embryos, hypocotyls, tubersegments, meristematic regions, and the like. The tissue source isselected and transformed so that it retains the ability to regeneratewhole, fertile plants following transformation, i.e., containstotipotent cells. Type I or Type II embryonic maize callus and immatureembryos are preferred Zea mays tissue sources. Similar tissues can betransformed for softwood or hardwood species. Selection of tissuesources for transformation of monocots is described in detail in U.S.application Ser. No. 08/112,245 and PCT publication WO 95/06128.

The transformation is carried out under conditions directed to the planttissue of choice. The plant cells or tissue are exposed to the DNA orRNA carrying the p-coumaroyl-CoA:monolignol transferase nucleic acidsfor an effective period of time. This may range from a less than onesecond pulse of electricity for electroporation to a 2-3 dayco-cultivation in the presence of plasmid-bearing Agrobacterium cells.Buffers and media used will also vary with the plant tissue source andtransformation protocol. Many transformation protocols employ a feederlayer of suspended culture cells (tobacco or Black Mexican Sweet corn,for example) on the surface of solid media plates, separated by asterile filter paper disk from the plant cells or tissues beingtransformed.

Electroporation:

Where one wishes to introduce DNA by means of electroporation, it iscontemplated that the method of Krzyzek et al. (U.S. Pat. No. 5,384,253)may be advantageous. In this method, certain cell wall-degradingenzymes, such as pectin-degrading enzymes, are employed to render thetarget recipient cells more susceptible to transformation byelectroporation than untreated cells. Alternatively, recipient cells canbe made more susceptible to transformation, by mechanical wounding.

To effect transformation by electroporation, one may employ eitherfriable tissues such as a suspension cell cultures, or embryogeniccallus, or alternatively, one may transform immature embryos or otherorganized tissues directly. The cell walls of the preselected cells ororgans can be partially degraded by exposing them to pectin-degradingenzymes (pectinases or pectolyases) or mechanically wounding them in acontrolled manner. Such cells would then be receptive to DNA uptake byelectroporation, which may be carried out at this stage, and transformedcells then identified by a suitable selection or screening protocoldependent on the nature of the newly incorporated DNA.

Microprojectile Bombardment:

A further advantageous method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method,microparticles may be coated with DNA and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

It is contemplated that in some instances DNA precipitation onto metalparticles would not be necessary for DNA delivery to a recipient cellusing microprojectile bombardment. In an illustrative embodiment,non-embryogenic BMS cells were bombarded with intact cells of thebacteria E. coli or Agrobacterium tumefaciens containing plasmids witheither the β-glucuronidase or bar gene engineered for expression inmaize. Bacteria were inactivated by ethanol dehydration prior tobombardment. A low level of transient expression of the 0-glucuronidasegene was observed 24-48 hours following DNA delivery. In addition,stable transformants containing the bar gene were recovered followingbombardment with either E. coli or Agrobacterium tumefaciens cells. Itis contemplated that particles may contain DNA rather than be coatedwith DNA. Hence it is proposed that particles may increase the level ofDNA delivery but are not, in and of themselves, necessary to introduceDNA into plant cells.

An advantage of microprojectile bombardment, in addition to it being aneffective means of reproducibly stably transforming monocots, is thatthe isolation of protoplasts (Christou et al., PNAS. 84:3962-3966(1987)), the formation of partially degraded cells, or thesusceptibility to Agrobacterium infection is not required. Anillustrative embodiment of a method for delivering DNA into maize cellsby acceleration is a Biolistics Particle Delivery System, which can beused to propel particles coated with DNA or cells through a screen, suchas a stainless steel or Nytex screen, onto a filter surface covered withmaize cells cultured in suspension (Gordon-Kamm et al., The Plant Cell.2:603-618 (1990)). The screen disperses the particles so that they arenot delivered to the recipient cells in large aggregates. It is believedthat a screen intervening between the projectile apparatus and the cellsto be bombarded reduces the size of projectile aggregate and maycontribute to a higher frequency of transformation, by reducing damageinflicted on the recipient cells by an aggregated projectile.

For bombardment, cells in suspension are preferably concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themacroprojectile stopping plate. If desired, one or more screens are alsopositioned between the acceleration device and the cells to bebombarded. Through the use of techniques set forth here-in one mayobtain up to 1000 or more foci of cells transiently expressing a markergene. The number of cells in a focus which express the exogenous geneproduct 48 hours post-bombardment often range from about 1 to 10 andaverage about 1 to 3.

In bombardment transformation, one may optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment can influence transformation frequency.Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the path andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells before andimmediately after bombardment, the osmotic adjustment of target cells tohelp alleviate the trauma associated with bombardment, and also thenature of the transforming DNA, such as linearized DNA or intactsupercoiled plasmid DNA.

One may wish to adjust various bombardment parameters in small scalestudies to fully optimize the conditions and/or to adjust physicalparameters such as gap distance, flight distance, tissue distance, andhelium pressure. One may also minimize the trauma reduction factors(TRFs) by modifying conditions which influence the physiological stateof the recipient cells and which may therefore influence transformationand integration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. Execution of such routineadjustments will be known to those of skill in the art.

An Example of Production and Characterization of Stable TransgenicMaize:

After effecting delivery of a p-coumaroyl-CoA:monolignol transferasenucleic acid to recipient cells by any of the methods discussed above,the transformed cells can be identified for further culturing and plantregeneration. As mentioned above, in order to improve the ability toidentify transformants, one may desire to employ a selectable orscreenable marker gene as, or in addition to, the expressiblep-coumaroyl-CoA:monolignol transferase nucleic acids. In this case, onewould then generally assay the potentially transformed cell populationby exposing the cells to a selective agent or agents, or one wouldscreen the cells for the desired marker gene trait.

Selection:

An exemplary embodiment of methods for identifying transformed cellsinvolves exposing the bombarded cultures to a selective agent, such as ametabolic inhibitor, an antibiotic, herbicide or the like. Cells whichhave been transformed and have stably integrated a marker geneconferring resistance to the selective agent used, will grow and dividein culture. Sensitive cells will not be amenable to further culturing.

To use the bar-bialaphos or the EPSPS-glyphosate selective system,bombarded tissue is cultured for about 0-28 days on nonselective mediumand subsequently transferred to medium containing from about 1-3 mg/lbialaphos or about 1-3 mM glyphosate, as appropriate. While ranges ofabout 1-3 mg/l bialaphos or about 1-3 mM glyphosate can be employed, itis proposed that ranges of at least about 0.1-50 mg/l bialaphos or atleast about 0.1-50 mM glyphosate will find utility in the practice ofthe invention. Tissue can be placed on any porous, inert, solid orsemi-solid support for bombardment, including but not limited to filtersand solid culture medium. Bialaphos and glyphosate are provided asexamples of agents suitable for selection of transformants, but thetechnique of this invention is not limited to them.

An example of a screenable marker trait is the red pigment producedunder the control of the R-locus in maize. This pigment may be detectedby culturing cells on a solid support containing nutrient media capableof supporting growth at this stage and selecting cells from colonies(visible aggregates of cells) that are pigmented. These cells may becultured further, either in suspension or on solid media. The R-locus isuseful for selection of transformants from bombarded immature embryos.In a similar fashion, the introduction of the C1 and B genes will resultin pigmented cells and/or tissues.

The enzyme luciferase is also useful as a screenable marker in thecontext of the present invention. In the presence of the substrateluciferin, cells expressing luciferase emit light which can be detectedon photographic or X-ray film, in a luminometer (or liquid scintillationcounter), by devices that enhance night vision, or by a highly lightsensitive video camera, such as a photon counting camera. All of theseassays are nondestructive and transformed cells may be cultured furtherfollowing identification. The photon counting camera is especiallyvaluable as it allows one to identify specific cells or groups of cellswhich are expressing luciferase and manipulate those in real time.

It is further contemplated that combinations of screenable andselectable markers may be useful for identification of transformedcells. For example, selection with a growth inhibiting compound, such asbialaphos or glyphosate at concentrations below those that cause 100%inhibition followed by screening of growing tissue for expression of ascreenable marker gene such as luciferase would allow one to recovertransformants from cell or tissue types that are not amenable toselection alone. In an illustrative embodiment embryogenic Type IIcallus of Zea mays L. can be selected with sub-lethal levels ofbialaphos. Slowly growing tissue was subsequently screened forexpression of the luciferase gene and transformants can be identified.

Regeneration and Seed Production:

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, are cultured in mediathat supports regeneration of plants. One example of a growth regulatorthat can be used for such purposes is dicamba or 2,4-D. However, othergrowth regulators may be employed, including NAA, NAA+2,4-D or perhapseven picloram. Media improvement in these and like ways can facilitatethe growth of cells at specific developmental stages. Tissue can bemaintained on a basic media with growth regulators until sufficienttissue is available to begin plant regeneration efforts, or followingrepeated rounds of manual selection, until the morphology of the tissueis suitable for regeneration, at least two weeks, then transferred tomedia conducive to maturation of embryoids. Cultures are typicallytransferred every two weeks on this medium. Shoot development signalsthe time to transfer to medium lacking growth regulators.

The transformed cells, identified by selection or screening and culturedin an appropriate medium that supports regeneration, can then be allowedto mature into plants. Developing plantlets are transferred to soillessplant growth mix, and hardened, e.g., in an environmentally controlledchamber at about 85% relative humidity, about 600 ppm CO₂, and at about25-250 microeinsteins/secm² of light. Plants can be matured either in agrowth chamber or greenhouse. Plants are regenerated from about 6 weeksto 10 months after a transformant is identified, depending on theinitial tissue. During regeneration, cells are grown on solid media intissue culture vessels. Illustrative embodiments of such vessels arepetri dishes and Plant Con™. Regenerating plants can be grown at about19° C. to 28° C. After the regenerating plants have reached the stage ofshoot and root development, they may be transferred to a greenhouse forfurther growth and testing.

Mature plants are then obtained from cell lines that are known toexpress the trait. In some embodiments, the regenerated plants areself-pollinated. In addition, pollen obtained from the regeneratedplants can be crossed to seed grown plants of agronomically importantinbred lines. In some cases, pollen from plants of these inbred lines isused to pollinate regenerated plants. The trait is geneticallycharacterized by evaluating the segregation of the trait in first andlater generation progeny. The heritability and expression in plants oftraits selected in tissue culture are of particular importance if thetraits are to be commercially useful.

Regenerated plants can be repeatedly crossed to inbred plants in orderto introgress the p-coumaroyl-CoA:monolignol transferase nucleic acidsinto the genome of the inbred plants. This process is referred to asbackcross conversion. When a sufficient number of crosses to therecurrent inbred parent have been completed in order to produce aproduct of the backcross conversion process that is substantiallyisogenic with the recurrent inbred parent except for the presence of theintroduced p-coumaroyl-CoA:monolignol transferase nucleic acids, theplant is self-pollinated at least once in order to produce a homozygousbackcross converted inbred containing the p-coumaroyl-CoA:monolignoltransferase nucleic acids. Progeny of these plants are true breeding.

Alternatively, seed from transformed monocot plants regenerated fromtransformed tissue cultures is grown in the field and self-pollinated togenerate true breeding plants.

Seed from the fertile transgenic plants can then be evaluated for thepresence and/or expression of the p-coumaroyl-CoA:monolignol transferasenucleic acids (or the p-coumaroyl-CoA:monolignol transferase enzyme).Transgenic plant and/or seed tissue can be analyzed forp-coumaroyl-CoA:monolignol transferase expression using standard methodssuch as SDS polyacrylamide gel electrophoresis, liquid chromatography(e.g., HPLC) or other means of detecting a product ofp-coumaroyl-CoA:monolignol transferase activity (e.g., coniferylcoumarate).

Once a transgenic seed expressing the p-coumaroyl-CoA:monolignoltransferase sequence and having an increase in monolignol coumarates inthe lignin of the plant is identified, the seed can be used to developtrue breeding plants. The true breeding plants are used to develop aline of plants with an increase in the percent of monolignol coumaratesin the lignin of the plant while still maintaining other desirablefunctional agronomic traits. Adding the trait of increased monolignolcoumarate production in the lignin of the plant can be accomplished byback-crossing with this trait and with plants that do not exhibit thistrait and studying the pattern of inheritance in segregatinggenerations. Those plants expressing the target trait in a dominantfashion are preferably selected. Back-crossing is carried out bycrossing the original fertile transgenic plants with a plant from aninbred line exhibiting desirable functional agronomic characteristicswhile not necessarily expressing the trait of an increased percent ofmonolignol coumarates in the lignin of the plant. The resulting progenyare then crossed back to the parent that expresses the increasedmonolignol coumarate trait. The progeny from this cross will alsosegregate so that some of the progeny carry the trait and some do not.This back-crossing is repeated until an inbred line with the desirablefunctional agronomic traits, and with expression of the trait involvingan increase in monolignol coumarates (e.g., coniferyl coumarate) withinthe lignin of the plant. Such expression of the increased percentage ofmonolignol coumarates in plant lignin can be expressed in a dominantfashion.

Subsequent to back-crossing, the new transgenic plants can be evaluatedfor an increase in the weight percent of monolignol coumaratesincorporated into the lignin of the plant. This can be done, forexample, by NMR analysis of whole plant cell walls (Kim, H., and Ralph,J. Solution-state 2D NMR of ball-milled plant cell wall gels inDMSO-d₆/pyridine-d₅. (2010) Org. Biomol. Chem. 8(3), 576-591; Yelle, D.J., Ralph, J., and Frihart, C. R. Characterization of non-derivatizedplant cell walls using high-resolution solution-state NMR spectroscopy.(2008) Magn. Reson. Chem. 46(6), 508-517; Kim, H., Ralph, J., andAkiyama, T. Solution-state 2D NMR of Ball-milled Plant Cell Wall Gels inDMSO-d₆. (2008) BioEnergy Research 1(1), 56-66; Lu, F., and Ralph, J.Non-degradative dissolution and acetylation of ball-milled plant cellwalls; high-resolution solution-state NMR. (2003) Plant J. 35(4),535-544). The new transgenic plants can also be evaluated for a batteryof functional agronomic characteristics such as lodging, kernelhardness, yield, resistance to disease, resistance to insect pests,drought resistance, and/or herbicide resistance.

Plants that may be improved by these methods include but are not limitedto oil and/or starch plants (canola, potatoes, lupins, sunflower andcottonseed), forage plants (alfalfa, clover and fescue), grains (maize,wheat, barley, oats, rice, sorghum, millet and rye), grasses(switchgrass, prairie grass, wheat grass, sudangrass, sorghum,straw-producing plants), softwood, hardwood and other woody plants(e.g., those used for paper production such as poplar species, pinespecies, and eucalyptus). In some embodiments the plant is a gymnosperm.Examples of plants useful for pulp and paper production include mostpine species such as loblolly pine, Jack pine, Southern pine, Radiatapine, spruce, Douglas fir and others. Hardwoods that can be modified asdescribed herein include aspen, poplar, eucalyptus, and others. Plantsuseful for making biofuels and ethanol include corn, grasses (e.g.,miscanthus, switchgrass, and the like), as well as trees such as poplar,aspen, willow, and the like. Plants useful for generating dairy forageinclude legumes such as alfalfa, as well as forage grasses such asbromegrass, and bluestem.

Determination of Stably Transformed Plant Tissues:

To confirm the presence of the p-coumaroyl-CoA:monolignol transferasenucleic acids in the regenerating plants, or seeds or progeny derivedfrom the regenerated plant, a variety of assays may be performed. Suchassays include, for example, molecular biological assays available tothose of skill in the art, such as Southern and Northern blotting andPCR; biochemical assays, such as detecting the presence of a proteinproduct, e.g., by immunological means (ELISAs and Western blots) or byenzymatic function; plant part assays, such as leaf, seed or rootassays; and also, by analyzing the phenotype of the whole regeneratedplant.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA may only be expressed in particular cells ortissue types and so RNA for analysis can be obtained from those tissues.PCR techniques may also be used for detection and quantification of RNAproduced from introduced p-coumaroyl-CoA:monolignol transferase nucleicacids. PCR also be used to reverse transcribe RNA into DNA, usingenzymes such as reverse transcriptase, and then this DNA can beamplified through the use of conventional PCR techniques. Furtherinformation about the nature of the RNA product may be obtained byNorthern blotting. This technique will demonstrate the presence of anRNA species and give information about the integrity of that RNA. Thepresence or absence of an RNA species can also be determined using dotor slot blot Northern hybridizations. These techniques are modificationsof Northern blotting and also demonstrate the presence or absence of anRNA species.

While Southern blotting and PCR may be used to detect thep-coumaroyl-CoA:monolignol transferase nucleic acid in question, they donot provide information as to whether the preselected DNA segment isbeing expressed. Expression may be evaluated by specifically identifyingthe protein products of the introduced p-coumaroyl-CoA:monolignoltransferase nucleic acids or evaluating the phenotypic changes broughtabout by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques such as ion exchange, liquid chromatography or gel exclusionchromatography. The unique structures of individual proteins offeropportunities for use of specific antibodies to detect their presence informats such as an ELISA assay. Combinations of approaches may beemployed with even greater specificity such as Western blotting in whichantibodies are used to locate individual gene products that have beenseparated by electrophoretic techniques. Additional techniques may beemployed to absolutely confirm the identity of thep-coumaroyl-CoA:monolignol transferase such as evaluation by amino acidsequencing following purification. The Examples of this application alsoprovide assay procedures for detecting and quantifyingp-coumaroyl-CoA:monolignol transferase activity. Other procedures may beadditionally used.

The expression of a gene product can also be determined by evaluatingthe phenotypic results of its expression. These assays also may takemany forms including but not limited to analyzing changes in thechemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of preselectedDNA segments encoding storage proteins which change amino acidcomposition and may be detected by amino acid analysis.

Definitions

As used herein, “isolated” means a nucleic acid or polypeptide has beenremoved from its natural or native cell. Thus, the nucleic acid orpolypeptide can be physically isolated from the cell or the nucleic acidor polypeptide can be present or maintained in another cell where it isnot naturally present or synthesized.

As used herein, a “native” nucleic acid or polypeptide means a DNA, RNAor amino acid sequence or segment that has not been manipulated invitro, i.e., has not been isolated, purified, and/or amplified.

EXAMPLES Example 1: Identification and Testing of Panicum virgatum andSorghum bicolor PMTs

This Example illustrates isolation and expression of enzymaticallyactive PMTs from Panicum virgatum and Sorghum bicolor.

Introduction

Recent work in plant biomass valorization has focused on the developmentof co-products to the production of cellulosic biofuels. One method ofvalorization is to increase the amount of easily clipped-off compoundsfor up-conversion to commodity chemicals. One such compound isp-coumaric acid (pCA), which can be found on the lignins of many monocotplants, including all grasses. Lignin is comprised predominantly ofmonolignols (ML) with three primary subunits: p-hydroxyphenyl (H),guaiacyl (G), and syringyl (S). The monolignols are known to formmonolignol p-coumarate conjugates (ML-pCA). These units are formed by aspecific subclass of BAHD acyl transferases known as p-coumaryl-coenzymeA monolignol transferases (PMTs). Introducing ML-pCAs into plants thatdon't originally have them has been shown also to improve ligninpretreatment and saccharification.

Methods

Selection of Gene Sequences

Gene sequences from sorghum, switchgrass, Brachypodium, maize, and ricewere identified from NCBI GenBank and Joint Genome Institute Phytozomeby their PFAM and BAHD transferase domain identity. Protein sequencecomparisons were made with NCBI BLASTP (blast.ncbi.nlm.nih.gov) usingdefault settings. The sequence identity is reported both as apercentage, as well as a fraction, where the numerator is the number ofidentical residues, and the denominator is the length of the matchedregion. See Table 1. The nucleic acid coding sequences and amino acidsequences encoded thereby for each of the Panicum virgatum (switchgrass)PMT (PvPMT) and the Sorghum bicolor (sorghum) PMT (SbPMT) are providedelsewhere herein. Each of the native genes for the PvPMT, and SbPMTproteins include introns, which are excluded from the sequences providedherein.

Cloning Vector

Coding sequences were synthesized by GenScript Corporation (Piscataway,N.J.) and cloned into the wheat germ cell-free expression vector, pEU(Sawasaki, T., Hasegawa, Y., Tsuchimochi, M., Kasahara, Y. and Endo, Y.(2000) Construction of an efficient expression vector for coupledtranscription/translation in a wheat germ cell-free system. NucleicAcids Symp Ser, 9-10), which contains an SP6 promoter and omega enhancersequence from tobacco mosaic virus. Plasmid DNA was purified from E.coli using a commercial purification kit, then treated with proteinase Kand re-purified to remove residual RNAse activity and to determineconcentration of the DNA.

Transcription

Messenger RNA was prepared by adding 1.6 U of SP6 RNA polymerase and 1 Uof RNasin RNase inhibitor (Promega Corporation, Madison, Wis.) toplasmid DNA (0.2 mg/mL or higher) in the presence of 2.5 mM each of UTP,CTP, ATP, and GTP and 20 mM magnesium acetate, 2 mM spermidine HCl, 10mM DTT, and 80 mM HEPES-KOH, pH 7.8. Transcription reactions wereincubated at 37° C. for 4 h and visually monitored for the appearance ofinsoluble pyrophosphate byproducts, which are indicative of successfultranscription.

Cell Free Translation

The active enzymes were produced using a wheat germ cell-freetranslation bilayer method previously reported (Makino, S., Beebe, E.T., Markley, J. L. and Fox, B. G. (2014) Cell-free protein synthesis forfunctional and structural studies. Methods in Molecular Biology, 1091,161-178). Briefly, a translation reaction mixture consisting of 60 ODwheat germ extract (CellFree Sciences, Matsuyama, Japan), 0.04 mg/mLcreatine kinase, 0.3 mM each amino acid, 12.6 mM HEPES-KOH, pH 7.8, 52.6mM potassium acetate, 1.3 mM magnesium acetate, 0.2 mM spermidine HCl,2.1 mM DTT, 0.6 mM ATP, 0.13 mM GTP, 8.4 mM creatine phosphate, and0.003% sodium azide was prepared and combined with non-purified, freshtranscription at a ratio of 4 parts reaction mix to 1 parttranscription. A feeding layer was prepared consisting of 0.3 mM eachamino acid, 24 mM HEPES-KOH, pH 7.8, 100 mM potassium acetate, 2.5 mMmagnesium acetate, 0.4 mM spermidine HCl, 4 mM DTT, 1.2 mM ATP, 0.25 mMGTP, 16 mM creatine phosphate, and 0.005% sodium azide, of which 125 aLwas added to wells of a U-bottom 96-well plate. 25 μL of the densertranslation reaction mixture was carefully underlayed below the feedinglayer, forming a bilayer. The plate was sealed and incubated at 22° C.for 18 h. The fully-diffused 150-μL bilayer reaction was then harvestedand used for expression analysis by SDS-PAGE, and activity screening.

Activity Screening

The enzyme mixture was screened for activity with p-coumaroyl-CoA (FIG.2B) and feruloyl-CoA (FIG. 2C) and all three monolignols (FIG. 2A)(p-coumaryl, coniferyl, and sinapyl alcohol). Each enzyme was testedindividually alongside positive and negative controls following theprocedure previously reported (Withers, S., Lu, F., Kim, H., Zhu, Y.,Ralph, J. and Wilkerson, C. G. (2012) Identification of a grass-specificenzyme that acylates monolignols with p-coumarate. Journal of BiologicalChemistry, 287, 8347-8355). Briefly, the assay was initiated by adding10 μL of wheat germ cell-free translation containing one of the PMTenzymes at a concentration of 1.5-2 μM to a reaction containing 50 mMsodium phosphate buffer, pH 6, 1 mM dithiothreitol (DTT), 1 mM CoAthioester, 1 mM monolignol mixture (each monolignol at 1 mMconcentration), and deionized water in a final volume of 50 μL. After a30-min incubation, the reaction was stopped by the addition of an equalvolume 100 mM hydrochloric acid. Reaction products were solubilized byadjusting the solution to 50% methanol. An identical assay with noenzyme added was performed for every reaction. Samples were filteredthrough 0.2 μm filters prior to analysis by liquid chromatography-massspectrometry (LC-MS).

Results

As shown in Table 1 and FIGS. 3A and 4A, both PvPMT and SbPMT are activePMTs, coupling monolignols to p-coumaroyl-CoA. These two enzymesincrease the known collection of PMTs to six, adding to ZmPMT/pCAT(GRMZM2G028104_P01) from maize (Table 1, FIG. 5), OsPMT (LOC_Os01g18744)from rice (Table 1, FIG. 6), and BdPMT1 (Bradi2g36910.1) and BdPMT2(Bradi1g36980.1) from Brachypodium (Table 1, FIG. 7). For a descriptionof OsPMT, BdPMT1, BdPMT2, and ZmPMT, see Withers et al. 2012 (Withers,S.; Lu, F.; Kim, H.; Zhu, Y.; Ralph, J.; Wilkerson, C. G.,Identification of a grass-specific enzyme that acylates monolignols withp-coumarate. J. Biol. Chem. 2012, 287 (11), 8347-8355), Petrik et al.2014 (Petrik, D. L.; Karlen, S. D.; Cass, C. L.; Padmakshan, D.; Lu, F.;Liu, S.; Le Bris, P.; Antelme, S.; Santoro, N.; Wilkerson, C. G.;Sibout, R.; Lapierre, C.; Ralph, J.; Sedbrook, J. C.,p-Coumaroyl-CoA:Monolignol Transferase (PMT) acts specifically in thelignin biosynthetic pathway in Brachypodium distachyon. The PlantJournal 2014, 77 (5), 713-726), Petrik et al. 2016 (Petrik, D. L.; Cass,C. L.; Padmakshan, D.; Foster, C. E.; Vogel, J. P.; Karlen, S. D.;Ralph, J.; Sedbrook, J. C., BdCESA7, BdCESA8, and BdPMT utility promoterconstructs for targeted expression to secondary cell-wall-forming cellsof grasses. Frontiers in Plant Science 2016, 7, 1-14), Sibout et al.2016 (Sibout, R.; Le Bris, P.; Legee, F.; Cezard, L.; Renault, H.;Lapierre, C., Structural redesigning Arabidopsis lignins intoalkali-soluble lignins through the expression ofp-coumaroyl-CoA:monolignol transferase PMT. Plant Physiol. 2016, 170(3), 1358-66), and Marita et al. 2014 (Marita, J. M.; Hatfield, R. D.;Rancour, D. M.; Frost, K. E., Identification and suppression of thep-coumaroyl CoA:hydroxycinnamyl alcohol transferase in Zea mays L. PlantJ. 2014, 78 (5), 850-864).

PvPMT and SbPMT have limited to no feruloyl-CoA monolignol transferaseactivity (Table 1 and FIGS. 3B and 4B). This finding is in contrast tothat of OsPMT1 which has been shown to have some feruloyl-CoA:monolignoltransferase (FMT) activity under the assay conditions (Withers, S.; Lu,F.; Kim, H.; Zhu, Y.; Ralph, J.; Wilkerson, C. G., Identification of agrass-specific enzyme that acylates monolignols with p-coumarate. J.Biol. Chem. 2012, 287 (11), 8347-8355).

TABLE 1 PMT enzymes similarity and activity. % Identity (SequenceCoverage) AA vs. vs. vs. vs. Activity with H, G, S Enzyme SpeciesAccession # length OsPMT ZmPMT BdPMT1 BdPMT2 pCA-CoA FA-CoA OsPMT Oryzasativa LOC_Os01g18744 440 100%  64% 62% 45% + + (440/440) (282/441)(280/452) (197/439) ZmPMT Zea mays GRMZM2G028104_P01 436 64% 100%  70%46% + + (283/441) (436/436) (306/439) (196/426) BdPMT1 BrachypodiumBradi2g36910.1 450 64% 70% 100%  43% + distachyon (271/421) (306/439)(450/450) (177/414) BdPMT2 Brachypodium Bradi1g36980.1 432 45% 46% 43%100%  + distachyon (197/439) (196/426) (177/414) (432/432) PvPMT PanicumPavir.J00672.1 428 62% 78% 70% 48% + − virgatum (274/439) (338/431)(311/444) (203/422) SbPMT Sorghum Sb09g002910.1 437 64% 85% 70% 45% + −bicolor (282/442) (374/438) (313/444) (194/427) H, p-hydroxycinnamylalcohol (p-coumaryl alcohol); G, coniferyl alcohol; S, sinapyl alcohol;pCA-CoA, p-coumaroyl-CoA; FA-CoA, feruloyl-CoA.

To confirm the different activity profiles for the PMT enzymes withregard to their ability to use feruloyl-CoA and p-coumaroyl-CoA and,specifically, which monolignol substrates are preferred, we used theJoint Genome Institute platform to synthesize and clone the genes, andthen expressed them in the wheat germ cell-free translation system inorder to test the activity of the enzymes. To the mixture of enzymesproduced from the cell-free system, which included putative PMT enzymesof interest, we added a mixture of all three lignin monomers (H, G, andS monolignols) and various CoA ester substrates, includingp-coumaroyl-CoA and feruloyl-CoA. The two putative PMT enzymes (SbPMTand PvPMT from sorghum and switchgrass, respectively) were found toproduce monolignol p-coumarate conjugates when fed monolignols andp-coumaroyl-CoA (FIG. 9). SbPMT and PvPMT showed little to no activitywhen fed monolignols and feruloyl-CoA, contrary to OsFMT (Karlen, S. D.,Zhang, C., Peck, M. L., Smith, R. A., Padmakshan, D., Helmich, K. E.,Free, H. C. A., Lee, S., Smith, B. G., Lu, F., Sedbrook, J. C., Sibout,R., Grabber, J. H., Runge, T. M., Mysore, K. S., Harris, P. J., Bartley,L. E. and Ralph, J. (2016) Monolignol ferulate conjugates are naturallyincorporated into plant lignins. Science Advances, 2, e1600393), whichwas used as a positive control for monolignol ferulate production (FIG.10).

Example 2: Analysis of in Planta Expression of Panicum virgatum andSorghum bicolor PMTS

The data in Example 1 indicate that the SbPMT and PvPMT enzymes functionas feruloyl-CoA monolignol transferases. The present example showsexpression and activity of these enzymes in planta.

Methods

Gateway cloning technology (Invitrogen) was used to generate constructsto express the SbPMT, and PvPMT genes in planta. The gateway constructsgenerated were: ProUBQ10: SbPMT-GFP and ProUBQ10: PvPMT-GFP (pUBC:GFP;(Grefen, C., Donald, N., Hashimoto, K., Kudla, J., Schumacher, K., &Blatt, M. R. (2010) The Plant Journal 64, 355-365)). The SbPMT plantexpression construct was introduced into Agrobacterium tumefaciensstrain GV3101 and transformed into Arabidopsis thaliana, ecotype Col-0,using the floral dip method (Clough, S. J. & Bent, A. F. (1998) PlantJournal 16, 735-743) to generate transgenic plants.

Transgenic seeds were sterilized with chlorine gas for 4 h prior toplating on half-strength Murashige and Skoog media (Sigma-Aldrich) with25 mg/L glufosinate-ammonium (Basta; Fisher) to select fortransformants. Seedlings were grown under long-day conditions (16 hlight, 8 h dark, 20° C.) for one week and then positive transformantswere screened for the presence of GFP. Whole seedlings were placed on aglass slide in water and examined for the presence of GFP using anepifluorescent microscope and a GFP excitation/emission filter set(488/525). Seedlings that showed resistance to Basta and strongfluorescence under the GFP filters were planted in soil and grown underlong-day conditions.

After 4-5 weeks, 2-3 small leaves were collected from each plant forgenomic DNA extraction and genotyping PCR. Briefly, the leaves wereground in Shorty extraction buffer (200 mM Tris-HCl, 250 mM NaCl, 25 mMNaEDTA, 0.5% SDS) and the samples were then centrifuged for 3 min. Thesupernatant was collected and, after the addition of isopropanol (300μL) to precipitate the DNA, centrifuged again. The DNA pellet was washedwith 70% ethanol (500 μL), centrifuged again, and then the pellets wereallowed to air dry. The DNA was re-suspended in 100 μL of TE buffer, pH8.0. Genotyping PCR using MangoTaq polymerase (Bioline) was performed toconfirm the presence of SbPMT in Arabidopsis. Primers were designed toamplify part of Actin2 (At3g18780) from Arabidopsis as a positivecontrol for DNA quality. The primers used to amplify the genes arelisted in Table 2. PCR cycling conditions were as follows: 94° C. 1 min,(94° C. 10 s, 48° C. 15 s, 72° C. 45 s)×32, 72° C. 5 min, then cooled to4° C.

TABLE 2 Primer sequences used for amplification ofcontrol (Actin 2) and transgenic SbPMT in Arabidopsis genotyping study.Gene Primers (5′-3′) Actin2-F CCAGAAGGATGCATATGTTGGTGA (SEQ ID NO: 6)Actin2-R GAGGAGCCTCGGTAAGAAGA (SEQ ID NO: 7) SbPMT-FATGGGCACAATCGATGATA (SEQ ID NO: 8) SbPMT-RAGCTGAGCAGGGCTG (SEQ ID NO: 9)

HPLC analysis was performed on a Shimadzu LCMS8040 equipped with aProminence LC20. The mobile phase was a binary gradient of acetonitrileand water, pumped at 0.7 mL/min through a Phemonenex Kinetex 5μ XB-C18,100 Å, 250×4.6 mm column (P/N: 00G-4605-E0) equipped with a guardcolumn. The LC program was initially held at 5% acetonitrile for 2 min,then ramped over 28 min to 100% acetonitrile, held there for 4 min andramped back over 1 min to 5% acetonitrile and held for 15 min. Thesamples were injected with an autoinjector onto the XB-C18 column andthe eluent then flowed through a PDA detector scanning from 250-400 nmand into the MS ionization source operating in DUIS (ESI/APCI) mode with2.5 L/min nebulizing gas, 15 L/min drying gas, 250° C. DL temperature,and 400° C. heat block. The MS scanned the ions in negative-ion modefrom 120-600 m/z. Elution times for the analytes are reported in Table3.

TABLE 3 Retention times for the monolignols and monolignol conjugates.Compound Retention time p-coumaryl alcohol 13.07 min coniferyl alcohol14.11 min sinapyl alcohol 14.51 min sinapyl p-coumarate 23.59 minconiferyl p-coumarate 23.79 min sinapyl ferulate 23.73 min coniferylferulate 23.94 minResults

The in vitro data shown in Example 1 strongly indicates that the SbPMTand PvPMT enzymes functioning specifically as p-coumaroyl-CoA monolignoltransferases. The present example shows the activity of these enzymes inplanta. The SbPMT gene was cloned into the pUBC-GFP destination vectorand transformed into Arabidopsis thaliana. The PvPMT gene can similarlybe cloned into the pUBC-GFP vector, and the construct can similarly betransformed into Arabidopsis. Arabidopsis was used in this study becauseit is a useful model organism when studying novel monolignol conjugates.As noted in Smith et al. (Smith, R. A., Gonzales-Vigil, E., Karlen, S.D., Park, J.-Y., Lu, F., Wilkerson, C. G., Samuels, L., Mansfield, S.D., & Ralph, J. (2015) Plant Physiology 169, 2992-3001), Arabidopsisdoes not produce detectable levels of monolignol p-coumarates, therebymaking the presence and production of monolignol p-coumarates throughthe putative PMT enzymes more apparent. The SbPMT gene was expressedunder the control of the Arabidopsis Ubiquitin 10 promoter and fused toa green fluorescent protein (GFP) at the C-terminus. The ubiquitouspromoter was because to achieve the highest expression level possible,with the assumption that it would lead to higher levels of theconjugates. The C-terminal GFP tag allows us to confirm that the PMTenzyme is being produced by the plant and also determine theintracellular location of the enzyme.

Seeds were plated on media containing Basta antibiotic to select forseedlings that contained the proUBQO-SbPMT-GFP construct. Beforeplanting, all seedlings were subjected to fluorescence microscopicanalysis to confirm the presence of GFP (as a proxy for the presence ofthe PMT enzyme). Following the development of a healthy rosette (4 weeksafter planting), 2-3 small leaves were dissected from each plant andused for genotyping analysis to confirm the presence of the PMT gene inthe plants. Plants that did not express the PMT gene were marked aswild-type and are be used as control plants during the chemical analysisof lignin. The genotyping analysis confirmed the SbPMT gene has beensuccessfully transformed into Arabidopsis. The SbPMT gene was able to beamplified from the genomic DNA of respective transgenic plants and wasnot present in wild-type plants. Together with the GFP screening, thesedata indicate that the transgene is present in the transgenicArabidopsis and that the protein is being expressed.

Transgenic plants continue to grow under long-day conditions untilsenescence, at which point the plants are harvested for chemicalanalysis. Wild-type and transgenic plant samples are ground and solventextracted to remove water-, ethanol-, and acetone-soluble compounds(basically isolating the plant cell wall). The ground, dried cell wallsamples are then be subjected to derivatization followed by reductivecleavage (DFRC) lignin analysis (Karlen, S. D., Zhang, C., Peck, M. L.,Smith, R. A., Padmakshan, D., Helmich, K. E., Free, H. C. A., Lee, S.,Smith, B. G., Lu, F., et al. (2016) Science Advances 2, e1600393:1600391-1600399; Lu, F. & Ralph, J. (1997) Journal of Agricultural andFood Chemistry 45, 2590-2592; Lu, F. & Ralph, J. (1999) Journal ofAgricultural and Food Chemistry 47, 1988-1992). This assay has beenshown to yield peaks that are diagnostic for not only the production ofmonolignol conjugates (monolignol ferulates and monolignolp-coumarates), but also their incorporation into the lignins oftransgenic Arabidopsis thaliana (Smith, R. A., Gonzales-Vigil, E.,Karlen, S. D., Park, J.-Y., Lu, F., Wilkerson, C. G., Samuels, L.,Mansfield, S. D., & Ralph, J. (2015) Plant Physiology 169, 2992-3001;Smith, R. A., Scheutz, M., Karlen, S. D., Bird, D., Tokunaga, N., Sato,Y., Mansfield, S. D., Ralph, J., & Samuels, A. L. (2017) PlantPhysiology 174, 1028-1036). DFRC analysis is expected to confirm thatthe SbPMT enzyme produced by the transgenic Arabidopsis thaliana plantshave the expected PMT activity in planta and that monolignolp-coumarates will be produced.

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby specifically incorporated by reference to the same extent asif it had been incorporated by reference in its entirety individually orset forth herein in its entirety. Applicants reserve the right tophysically incorporate into this specification any and all materials andinformation from any such cited patents or publications.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, from 5to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and the methods and processes are not necessarilyrestricted to the orders of steps indicated herein or in the claims. Asused herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a nucleic acid” or “apolypeptide” includes a plurality of such nucleic acids or polypeptides(for example, a solution of nucleic acids or polypeptides or a series ofnucleic acid or polypeptide preparations), and so forth. Under nocircumstances may the patent be interpreted to be limited to thespecific examples or embodiments or methods specifically disclosedherein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official oremployee of the Patent and Trademark Office unless such statement isspecifically and without qualification or reservation expressly adoptedin a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims and statements of theinvention.

The following statements of the invention are intended to summarize someaspects of the invention according to the foregoing description given inthe specification.

STATEMENTS OF A FIRST SET OF EMBODIMENTS OF THE INVENTION

1. An isolated nucleic acid encoding a p-coumaroyl-CoA:monolignoltransferase wherein the nucleic acid can selectively hybridize to a DNAwith a SEQ ID NO:1 or SEQ ID NO:3 sequence.

2. The isolated nucleic acid of statement 1, wherein the nucleic acidselectively hybridizes to a DNA with a SEQ ID NO:1 or SEQ ID NO:3sequence under stringent hybridization conditions.

3. The isolated nucleic acid of statement 2, wherein the stringenthybridization conditions comprise a wash in 0.1×SSC, 0.1% SDS at 65° C.

4. The isolated nucleic acid of any of statements 1-3, wherein thenucleic acid that selectively hybridizes to a DNA with a SEQ ID NO:1 orSEQ ID NO:3 sequence has at least about 70% sequence identity with SEQID NO:1 or SEQ ID NO:3.

5. The isolated nucleic acid of any of statements 1-4, wherein thenucleic acid encodes a p-coumaroyl-CoA:monolignol transferase that cancatalyze the synthesis of monolignol coumarate(s) from monolignol(s) andcoumaroyl-CoA.

6. The isolated nucleic acid of statement 5, wherein the monolignol isconiferyl alcohol, p-coumaryl alcohol, sinapyl alcohol or a combinationthereof.

7. The isolated nucleic acid of any of statements 1-6, wherein thenucleic acid encodes a p-coumaroyl-CoA:monolignol transferasepolypeptide with a SEQ ID NO:2 or SEQ ID NO:4 sequence.

8. The isolated nucleic acid of any of statements 1-7, wherein thenucleic acid encodes a p-coumaroyl-CoA:monolignol transferase that cancatalyze the synthesis of monolignol coumarate(s) from a monolignol(s)and coumaroyl-CoA with at least about 50% of the activity of ap-coumaroyl-CoA:monolignol transferase with the SEQ ID NO:2 or SEQ IDNO:4.

9. A transgenic plant cell comprising the isolated nucleic acid of anyof statements 1-8.

10. A transgenic plant comprising the plant cell of statement 9 or theisolated nucleic acid of any of statements 1-8.

11. An expression cassette comprising the p-coumaroyl-CoA:monolignoltransferase nucleic acid of any of statements 1-8 operably linked to apromoter functional in a host cell.

12. The expression cassette of statement 11, which further comprises aselectable marker gene.

13. The expression cassette of statement 11 or 12, further comprisingplasmid DNA.

14. The expression cassette of any of statements 11-13, wherein theexpression cassette is within an expression vector.

15. The expression cassette of any of statements 11-14, wherein thepromoter is a promoter functional during plant development or growth.

16. The expression cassette of any of statements 11-15, wherein thepromoter is a poplar xylem-specific secondary cell wall specificcellulose synthase 8 promoter, cauliflower mosaic virus promoter, Z10promoter from a gene encoding a 10 kD zein protein, Z27 promoter from agene encoding a 27 kD zein protein, pea rbcS gene or actin promoter fromrice.

17. A plant cell comprising the expression cassette of any of statements11-16.

18. The plant cell of statement 17, wherein the plant cell is a monocotcell.

19. The plant cell of statement 17, wherein the plant cell is a maize,grass or softwood cell.

20. The plant cell of statement 17, wherein the plant cell is a dicotcell.

21. The plant cell of statement 17, wherein the plant cell is a hardwoodcell.

22. A plant comprising the expression cassette of any of statements11-16.

23. The plant of statement 22, wherein the plant is a monocot.

24. The plant of statement 22, wherein the plant is a grass, maize orsoftwood.

25. The plant of statement 22, wherein the plant is a gymnosperm.

26. The plant of statement 22, wherein the plant is a dicot.

27. The plant of statement 22, wherein the dicot is a hardwood.

28. A method for incorporating monolignol coumarates into lignin of aplant, comprising:

a) stably transforming plant cells with the expression cassette of anyof statements 11-16 to generate transformed plant cells;

b) regenerating the transformed plant cells into at least one transgenicplant, wherein p-coumaroyl-CoA:monolignol transferase is expressed in atleast one transgenic plant in an amount sufficient to incorporatemonolignol coumarates into the lignin of the transgenic plant.

29. The method of statement 28, wherein the transgenic plant is fertile.

30. The method of statement 28 or 29, further comprising recoveringtransgenic seeds from the transgenic plant, wherein the transgenic seedscomprise the nucleic acid encoding a p-coumaroyl-CoA:monolignoltransferase.

31. The method of any of statements 28-30, wherein the plant is amonocot.

32. The method of any of statements 28-31, wherein the plant is a grass,maize or softwood plant.

33. The method of any of statements 28-32, wherein the plant is agymnosperm.

34. The method of statement 28, wherein the plant is a dicot.

35. The method of statement 34, wherein the dicot plant is a hardwood.

36. The method of any of statements 28-35, wherein the lignin in theplant comprises at least 1% monolignol coumarate.

37. The method of any of statements 28-36, wherein the lignin in theplant comprises at least 5% monolignol coumarate.

38. The method of any of statements 28-37, wherein the lignin in theplant comprises at least 10% monolignol coumarate.

39. The method of any of statements 28-38, wherein the lignin in theplant comprises at least 20% monolignol coumarate.

40. The method of any of statements 28-39, wherein the lignin in theplant comprises at least 25% monolignol coumarate.

41. The method of any of statements 28-40, wherein the lignin in theplant comprises about 1-30% monolignol coumarate, or about 2-30%monolignol coumarate.

42. The method of any of statements 28-41, further comprising breeding afertile transgenic plant to yield a progeny plant that has an increasein the percentage of monolignol coumarates in the lignin of the progenyplant relative to the corresponding untransformed plant.

43. The method of any of statements 28-42, further comprising breeding afertile transgenic plant to yield a progeny plant that has an increasein the percentage of monolignol coumarates in the lignin of the progenyplant as a dominant trait while still maintaining functional agronomiccharacteristics relative to the corresponding untransformed plant.

44. The method of any of statements 28-43, wherein the transformed plantcell is transformed by a method selected from the group consisting ofelectroporation, microinjection, microprojectile bombardment, andliposomal encapsulation.

45. The method of any of statements 28-44, further comprising stablytransforming the plant cell with at least one selectable marker gene.

46. A fertile transgenic plant having an increased percent of monolignolcoumarates in the plant's lignin, the genome of which is stablytransformed by the nucleic acid of any of statements 1-8, wherein thenucleic acid is operably linked to a promoter functional in a host cell,and wherein the p-coumaroyl-CoA:monolignol transferase nucleic acid istransmitted through a complete normal sexual cycle of the transgenicplant to the next generation.

47. The plant of statement 46, wherein the plant is a monocot.

48. The plant of statement 46, wherein the plant is a grass, maize orsoftwood.

49. The plant of statement 46, wherein the plant is a gymnosperm.

50. The plant of statement 46, wherein the plant is a dicot.

51. The plant of statement 46, wherein the percent of monolignolcoumarates in the plant's lignin is increased relative to thecorresponding untransformed plant.

52. The plant of any of statements 46-51, wherein the percent ofmonolignol coumarates in the plant's lignin is increased by at least 1%relative to the corresponding untransformed plant.

53. The plant of any of statements 46-52, wherein the percent ofmonolignol coumarates in the plant's lignin is increased by at least2-5% relative to the corresponding untransformed plant.

54. The plant of any of statements 46-53, wherein the lignin in theplant comprises at least 1% monolignol coumarates.

55. The plant of any of statements 46-54, wherein the lignin in theplant comprises at least 5% monolignol coumarates.

56. The plant of any of statements 46-55, wherein the lignin in theplant comprises at least 10% monolignol coumarates.

57. The plant of any of statements 46-56, wherein the lignin in theplant comprises at least 20% monolignol coumarates.

58. The plant of any of statements 46-57, wherein the lignin in theplant comprises at least 25% monolignol coumarates.

59. The plant of any of statements 46-58, wherein the lignin in theplant comprises about 1-30% monolignol coumarates.

60. A lignin isolated from a transgenic plant comprising the isolatednucleic of any of statements 1-8.

61. A method of making a product from a transgenic plant comprising:

(a) providing or obtaining a transgenic plant that includes an isolatednucleic acid encoding a p-coumaroyl-CoA:monolignol transferasecomprising the isolated nucleic of any of statements 1-8; and

(b) processing the transgenic plant's tissues under conditionssufficient to digest the lignin; and thereby generate the product fromthe transgenic plant,

wherein the transgenic plant's tissues comprise lignin having anincreased percent of monolignol coumarates relative to a correspondinguntransformed plant.

62. The method of statement 61, wherein the conditions sufficient todigest the lignin comprise conditions sufficient to cleave ester bondswithin monolignol coumarate-containing lignin.

63. The method of statement 61 or 62, wherein the conditions sufficientto digest the lignin comprise mildly alkaline conditions.

64. The method of any of statements 61-63, wherein the conditionssufficient to digest the lignin comprise contacting the transgenicplant's tissues with ammonia for a time and a temperature sufficient tocleave ester bonds within monolignol coumarate-containing lignin.

65. The method of any of statements 61-64, wherein the conditionssufficient to digest the lignin would not cleave substantially any ofthe ether and carbon-carbon bonds in lignin from a corresponding plantthat does not contain the isolated nucleic acid encoding thep-coumaroyl-CoA:monolignol transferase.

STATEMENTS OF A SECOND SET OF EMBODIMENTS OF THE INVENTION

1A. A transgenic plant comprising a knockdown or knockout of the plant'sendogenous p-coumaroyl-CoA:monolignol transferase gene.

2A. The transgenic plant of statement 1A, further comprising aferuloyl-CoA:monolignol transferase nucleic acid operably linked to apromoter functional in cells of the transgenic plant.

3A. The transgenic plant of statement 1A, wherein the endogenousp-coumaroyl-CoA:monolignol transferase gene can hybridize to a nucleicacid with a sequence selected from the group consisting of SEQ ID NO:1and 3.

4A. The transgenic plant of statement 1A, wherein the endogenousp-coumaroyl-CoA:monolignol transferase gene has at least 50% sequenceidentity with a nucleic acid sequence selected from the group consistingof SEQ ID NO: 1 and 3.

5A. The transgenic plant of statement 1A, wherein the knockdown orknockout is a mutation selected from the group consisting of a pointmutation, a deletion, a missense mutation, insertion or a nonsensemutation in the endogenous p-coumaroyl-CoA:monolignol transferase gene.

6A. The transgenic plant of statement 1A, wherein the knockdown orknockout mutation comprises a point mutation, a deletion, a missensemutation, insertion or a nonsense mutation in the endogenousp-coumaroyl-CoA:monolignol transferase gene encoding a polypeptide withat least 60% sequence identity to an amino acid sequence selected fromthe group consisting of SEQ ID NO: 2 and 4.

7A. The transgenic plant of statement 1A, wherein expression of at leastone inhibitory nucleic acid comprising a nucleic acid sequence with atleast 90% sequence identity to either strand of a nucleic acidcomprising a sequence selected from the group consisting of SEQ ID NO: 1and 3 comprises the knockdown or knockout.

8A. The transgenic plant of statement 1A, wherein the knockdown orknockout reduces acylation of monolignols with p-coumarate.

9A. The transgenic plant of statement 1A, wherein the knockdown orknockout reduces acylation of monolignols with p-coumarate, where themonolignols are selected from the group consisting of p-coumarylalcohol, coniferyl alcohol and sinapyl alcohol.

10A. The transgenic plant of statement 1A, wherein the knockdown orknockout reduces acylation of monolignols with p-coumarate by at leastby 30%.

11A. The transgenic plant of statement 2A, wherein theferuloyl-CoA:monolignol transferase nucleic acid encodes an amino acidsequence of any feruloyl-CoA:monolignol transferase sequenceincorporated herein by reference.

12A. The transgenic plant of statement 2A, wherein theferuloyl-CoA:monolignol transferase nucleic acid is operably linked to apromoter selected from the group consisting of a poplar xylem-specificsecondary cell wall specific cellulose synthase 8 promoter, cauliflowermosaic virus promoter, Z10 promoter from a gene encoding a 10 kD zeinprotein, Z27 promoter from a gene encoding a 27 kD zein protein, pearbcS gene, or anactin promoter from rice.

13A. The transgenic plant of statement 1A, wherein the plant is a grassspecies.

14A. The transgenic plant of statement 1A, wherein the plant is selectedfrom the species consisting of Miscanthus giganteus, Panicum virgatum(switchgrass), Zea mays (corn), Oryza sativa (rice), Saccharum sp.(sugar cane), Triticum sp. (wheat), Avena sativa (oats), Pennisetumglaucum (pearl millet), Setaria italica (foxtail millet), Sorghum sp.(e.g., Sorghum bicolor), Bambuseae species (bamboo), Sorghastrum nutans(indiangrass), Tripsacum dactyloides (eastern gamagrass), Andropogongerardii (big bluestem), Schizachyrium scoparium (little bluestem),Bouteloua curtipendula (sideoats grama), Silphium terebinthinaceum(prairie rosinweed), Pseudoroegneria spicata (bluebunch wheatgrass)Sorghum bicolor (sorghum) and Bachypodium distachyon (purple falsebrome).

15A. The transgenic plant of statement 1A, wherein the plant is fertile.

16A. One or more seeds from the transgenic plant of statement 1A.

17A. An inhibitory nucleic acid comprising a DNA or RNA comprising anucleic acid sequence with at least 90% sequence identity to eitherstrand of a nucleic acid comprising a sequence selected from the groupconsisting of SEQ ID NO:1 and 3.

18A. An expression cassette comprising the inhibitory nucleic acid ofstatement 17A operably linked to a promoter functional in a host cell.

19A. An isolated cell comprising the inhibitory nucleic acid ofstatement 17A or the expression cassette of statement 18A.

20A. The isolated cell of statement 19A, which is a microorganism or aplant cell.

21A. The isolated cell of statement 19A, wherein the cell is a grassplant cell.

22A. The isolated cell of statement 19A, wherein the cell is a plantcell selected from the species consisting of Miscanthus giganteus,Panicum virgatum (switchgrass), Zea mays (corn), Oryza sativa (rice),Saccharum sp. (sugar cane), Triticum sp. (wheat), Avena sativa (oats),Pennisetum glaucum (pearl millet), Setaria italica (foxtail millet),Sorghum sp. (e.g., Sorghum bicolor), Bambuseae species (bamboo),Sorghastrum nutans (indiangrass), Tripsacum dactyloides (easterngamagrass), Andropogon gerardii (big bluestem), Schizachyrium scoparium(little bluestem), Bouteloua curtipendula (sideoats grama), Silphiumterebinthinaceum (prairie rosinweed), Pseudoroegneria spicata (bluebunchwheatgrass) Sorghum bicolor (sorghum) and Bachypodium distachyon (purplefalse brome).

23A. A transgenic plant comprising the isolated cell of statement 19A.

24A. A method of incorporating monolignol ferulates into lignin of aplant comprising: a) obtaining one or more plant cells having a knockoutor knockdown of the plant cells' endogenous p-coumaroyl-CoA:monolignoltransferase gene; b) regenerating one or more of the plant cells into atleast one transgenic plant.

25A. The method of statement 24A, further comprising stably transformingthe one or more plant cells with an expression cassette comprising aferuloyl-CoA:monolignol transferase nucleic acid operably linked to apromoter to generate one or more transformed plant cells with theendogenous p-coumaroyl-CoA:monolignol transferase knockout or knockdownmutation, before regenerating the cells into at least one transgenicplant.

26A. A method of incorporating monolignol ferulates into lignin of aplant comprising: a) obtaining one or more plant cells stablytransformed with a feruloyl-CoA:monolignol transferase nucleic acidoperably linked to a promoter to generate at least one transformed plantcell; b) mutating the at least transformed plant cell to generate atleast one transformed mutant plant cell with a knockout or knockdownmutation of the plant cell's endogenous p-coumaroyl-CoA:monolignoltransferase gene; c) regenerating one or more of the transformed mutantplant cells into at least one transgenic plant.

27A. The method of statement 24A, wherein the endogenousp-coumaroyl-CoA:monolignol transferase gene can hybridize to a nucleicacid with a sequence selected from the group consisting of SEQ ID NO:1and 3.

28A. The method of statement 24A, wherein the endogenousp-coumaroyl-CoA:monolignol transferase gene has at least 50% sequenceidentity, with a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1 and 3.

29A. The method of statement 24A, wherein the knockdown or knockoutcomprises a point mutation, a deletion, a missense mutation, aninsertion or a nonsense mutation in the endogenousp-coumaroyl-CoA:monolignol transferase gene.

30A. The method of statement 24A, wherein the knockdown or knockoutmutation comprises a point mutation, a deletion, a missense mutation,insertion or a nonsense mutation in the endogenousp-coumaroyl-CoA:monolignol transferase gene encoding a polypeptide withat least 60% sequence identity to an amino acid sequence selected fromthe group consisting of SEQ ID NO:2 and 4.

31A. The method of statement 24A, wherein expression of at least oneinhibitory nucleic acid comprising a nucleic acid sequence with at least90% sequence identity to either strand of a nucleic acid comprising asequence selected from the group consisting of SEQ ID NO: 1 and 3comprises the knockdown or knockout.

32A. The method of statement 24A, wherein the knockdown or knockoutreduces acylation of monolignols with p-coumarate.

33A. The method of statement 24A, wherein the knockdown or knockoutreduces acylation of monolignols with p-coumarate, where the monolignolsare selected from the group consisting of p-coumaryl alcohol, coniferylalcohol and sinapyl alcohol.

34A. The method of statement 24A, wherein the knockdown or knockoutreduces acylation of monolignols with p-coumarate by at least by 30%.

35A. The method of statement 24A, wherein the feruloyl-CoA:monolignoltransferase nucleic acid encodes an amino acid sequence selected fromthe group consisting of any feruloyl-CoA:monolignol transferase sequenceincorporated herein by reference, or an amino acid sequence with atleast 60% sequence identity to an amino acid sequence selected from thegroup consisting of any feruloyl-CoA:monolignol transferase sequenceincorporated herein by reference.

36A. The method of statement 25A, wherein the feruloyl-CoA:monolignoltransferase nucleic acid is operably linked to a promoter selected fromthe group consisting of a poplar xylem-specific secondary cell wallspecific cellulose synthase 8 promoter, cauliflower mosaic viruspromoter, Z10 promoter from a gene encoding a 10 kD zein protein, Z27promoter from a gene encoding a 27 kD zein protein, pea rbcS gene, oranactin promoter from rice.

37A. The method of statement 24A, wherein the plant is a grass species.

38A. The method of statement 24A, wherein the plant is selected from thespecies consisting of Miscanthus giganteus, Panicum virgatum(switchgrass), Zea mays (corn), Oryza sativa (rice), Saccharum sp.(sugar cane), Triticum sp. (wheat), Avena sativa (oats), Pennisetumglaucum (pearl millet), Setaria italica (foxtail millet), Sorghum sp.(e.g., Sorghum bicolor), Bambuseae species (bamboo), Sorghastrum nutans(indiangrass), Tripsacum dactyloides (eastern gamagrass), Andropogongerardii (big bluestem), Schizachyrium scoparium (little bluestem),Bouteloua curtipendula (sideoats grama), Silphium terebinthinaceum(prairie rosinweed), Pseudoroegneria spicata (bluebunch wheatgrass)Sorghum bicolor (sorghum) and Bachypodium distachyon (purple falsebrome).

39A. The method of statement 24A, wherein the plant is fertile.

40A. The method of statement 24A, further comprising isolating seedsfrom the plant.

41A. A method of inhibiting expression and/or translation ofp-coumaroyl-CoA:monolignol transferase RNA in a plant cell comprising:a) contacting or transforming plant cells with the expression cassetteof statement 18A to generate transformed plant cells; b) regeneratingthe transformed plant cells into at least one transgenic plant, whereinan inhibitory nucleic acid adapted to inhibit the expression and/ortranslation of a p-coumaroyl-CoA:monolignol transferase mRNA isexpressed in at least one transgenic plant in an amount sufficient toincorporate monolignol ferulates into the lignin of the transgenicplant.

42A. The method of statement 41A, wherein the plant cells are stablytransformed with a feruloyl-CoA:monolignol transferase nucleic acidoperably linked to a promoter.

STATEMENTS OF A THIRD SET OF EMBODIMENTS OF THE INVENTION

1B. An isolated nucleic acid encoding at least a portion of ap-coumaroyl-CoA:monolignol transferase, and/or an isolated nucleic acidcomplementary to at least a portion of a p-coumaroyl-CoA:monolignoltransferase nucleic acid, wherein the isolated nucleic acid canselectively hybridize to a DNA or RNA with a sequence homologous orcomplementary to a sequence selected from the group consisting of SEQ IDNO: 1 and 3, and a combination thereof.

2B. The isolated nucleic acid of statement 1B, wherein the nucleic acidselectively hybridizes to a DNA or RNA comprising either strand of anyof the SEQ ID NO: 1 and 3 sequences under physiological conditionswithin a live plant cell.

3B. The isolated nucleic acid of statement 1B, wherein the nucleic acidselectively hybridizes to a DNA or RNA comprising either strand of anyof the SEQ ID NO: 1 and 3 sequences under stringent hybridizationconditions.

4B. The isolated nucleic acid of statement 3B, wherein the stringenthybridization conditions comprise a wash in 0.1×SSC, 0.1% SDS at 65° C.

5B. The isolated nucleic acid of any of statements 1B-5B, wherein thenucleic acid that selectively hybridizes to a DNA or RNA has at leastabout 40%, 50%, 60%, 70%, 80%, 90% sequence identity with either strandof any of the SEQ ID NO: 1 and 3 sequences.

6B. The isolated nucleic acid of any of statements 1B-5B, wherein thenucleic acid encodes a p-coumaroyl-CoA:monolignol transferase that cancatalyze the synthesis of monolignol p-coumarate(s) from monolignol(s)and p-coumaroyl-CoA.

7B. The isolated nucleic acid of statement 6B, wherein the monolignol isconiferyl alcohol, p-coumaryl alcohol, sinapyl alcohol or a combinationthereof.

8B. The isolated nucleic acid of any of statements 1B-7B, wherein thenucleic acid encodes a polypeptide with at least 50%, 60%, 70%, 80%, or90% sequence identity to a polypeptide comprising a SEQ ID NO:2 or SEQID NO:4 sequence.

9B. The isolated nucleic acid of any of statements 1B-8B, wherein thenucleic acid encodes p-coumaroyl-CoA:monolignol transferase that cancatalyze the synthesis of monolignol p-coumarate(s) from a monolignol(s)and p-coumaroyl-CoA with at least about 50% of the activity of ap-coumaroyl-CoA:monolignol transferase with the SEQ ID NO:2 or SEQ IDNO:4.

10B. The isolated nucleic acid of any of statements 1B-9B, where theisolated nucleic acid is an inhibitory nucleic acid adapted to inhibitthe expression and/or translation of a p-coumaroyl-CoA:monolignoltransferase mRNA.

11B. The isolated nucleic acid of any of statements 1B-9B, where theisolated nucleic acid is mutating nucleic acid that binds to anendogenous p-coumaroyl-CoA:monolignol transferase gene in a cell ofgrass species.

12B. The isolated nucleic acid of statement 11B, wherein the mutatingnucleic has two flanking segments and a central segment,

wherein the central segment has a point mutation, a deletion, a missensemutation, or a nonsense mutation relative to a nucleic acid selectedfrom the group consisting of SEQ ID NO: 1 and SEQ ID NO:3; and

wherein the two flanking segments are separately homologous orcomplementary to a different region of a nucleic acid selected from thegroup consisting of SEQ ID NO: 1 and SEQ ID NO:3.

13B. A transgenic plant cell comprising the isolated nucleic acid of anyof statements 1B-12B.

14B. A transgenic plant comprising the plant cell of statement 12B orthe isolated nucleic acid of any of statements 1B-13B.

15B. An expression cassette comprising the p-coumaroyl-CoA:monolignoltransferase nucleic acid of any of statements 1B-14B operably linked toa promoter functional in a host cell.

16B. The expression cassette of statement 15B, further comprising aferuloyl-CoA:monolignol transferase nucleic acid operably linked to apromoter functional in a host cell.

17B. The expression cassette of statement 15B or 16B, which furthercomprises a selectable marker gene.

18B. The expression cassette of any of statements 15B-17B, wherein theexpression cassette is within an expression vector.

19B. The expression cassette of any of statements 15B-18B, wherein atleast one of the promoters is a promoter functional during plantdevelopment or growth.

20B. The expression cassette of any of statements 15B-19B, wherein atleast one of the promoters is a poplar xylem-specific secondary cellwall specific cellulose synthase 8 promoter, cauliflower mosaic viruspromoter, Z10 promoter from a gene encoding a 10 kD zein protein, Z27promoter from a gene encoding a 27 kD zein protein, pea rbcS gene oractin promoter from rice.

21B. A plant cell comprising the expression cassette of any ofstatements 15B-20B.

22B. The plant cell of statement 21B, wherein the plant cell is amonocot cell, maize cell, grass cell or softwood cell.

23B. The plant cell of statement 21B or 22B, wherein the plant cell is acell selected from the species consisting of Miscanthus giganteus,Panicum virgatum (switchgrass), Zea mays (corn), Oryza sativa (rice),Saccharum sp. (sugar cane), Triticum sp. (wheat), Avena sativa (oats),Pennisetum glaucum (pearl millet), Setaria italica (foxtail millet),Sorghum sp. (e.g., Sorghum bicolor), Bambuseae species (bamboo),Sorghastrum nutans (indiangrass), Tripsacum dactyloides (easterngamagrass), Andropogon gerardii (big bluestem), Schizachyrium scoparium(little bluestem), Bouteloua curtipendula (sideoats grama), Silphiumterebinthinaceum (prairie rosinweed), Pseudoroegneria spicata (bluebunchwheatgrass) Sorghum bicolor (sorghum) and Bachypodium distachyon (purplefalse brome).

24B. The plant cell of statement 21B, wherein the plant cell is a dicotcell or a hardwood cell.

25B. A plant comprising the expression cassette of any of statements15B-20B.

26B. The plant of statement 25B, wherein the plant is a monocot such asa grass species.

27B. The plant of statement 25B or 26B, wherein the plant is selectedfrom the species consisting of Miscanthus giganteus, Panicum virgatum(switchgrass), Zea mays (corn), Oryza sativa (rice), Saccharum sp.(sugar cane), Triticum sp. (wheat), Avena sativa (oats), Pennisetumglaucum (pearl millet), Setaria italica (foxtail millet), Sorghum sp.(e.g., Sorghum bicolor), Bambuseae species (bamboo), Sorghastrum nutans(indiangrass), Tripsacum dactyloides (eastern gamagrass), Andropogongerardii (big bluestem), Schizachyrium scoparium (little bluestem),Bouteloua curtipendula (sideoats grama), Silphium terebinthinaceum(prairie rosinweed), Pseudoroegneria spicata (bluebunch wheatgrass)Sorghum bicolor (sorghum) and Bachypodium distachyon (purple falsebrome).

28B. The plant of statement 25B, wherein the plant is a dicot or ahardwood.

29B. A method for incorporating monolignol ferulates into lignin of aplant comprising:

a) obtaining one or more plant cells having a knockout or knockdownmutation of the plant cells' endogenous p-coumaroyl-CoA:monolignoltransferase gene;

b) stably transforming the one or more plant cells with an expressioncassette comprising a feruloyl-CoA:monolignol transferase nucleic acidto generate one or more transformed plant cells with the endogenousp-coumaroyl-CoA:monolignol transferase knockout or knockdown mutation;

c) regenerating one or more of the transformed plant cells into at leastone transgenic plant.

30B. The method of statement 29B, wherein the knockout or knockdownmutation increases incorporation of monolignol ferulates into the ligninof at least one of the transgenic plants compared to a control plantthat (a) does not have the knockout or knockdown mutation but (b) isstably transformed with the expression cassette comprisingferuloyl-CoA:monolignol transferase nucleic acid.

31B. The method of statement 29B or 30B, wherein the knockout orknockdown mutation increases incorporation of monolignol ferulates intothe lignin of a plant by at least by 1%, or by at least 2%, or by atleast 3%, or by at least 5% relative to a control plant that (a) doesnot have the knockout or knockdown mutation but (b) is stablytransformed with the expression cassette comprisingferuloyl-CoA:monolignol transferase nucleic acid.

32B. The method of any of statements 29B-31B, wherein the endogenousp-coumaroyl-CoA:monolignol transferase gene can hybridize to a nucleicacid selected from the group consisting of SEQ ID NO:1 and 3; or theendogenous p-coumaroyl-CoA:monolignol transferase gene has at least 40%sequence identity, at least 45% sequence identity, at least 50% sequenceidentity, at least 55% sequence identity, at least 60% sequenceidentity, at least 65% sequence identity, at least 70% sequenceidentity, at least 75% sequence identity, at least 80% sequenceidentity, at least 85% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, or at least 97% sequenceidentity with a nucleic acid sequence selected from the group consistingof SEQ ID NO:1 and 3.

33B. A method for incorporating monolignol ferulates into lignin of aplant that includes:

a) stably transforming one or more plant cells with a mutating nucleicacid adapted to hybridize to an endogenous p-coumaroyl-CoA:monolignoltransferase gene within the plant cells and replace at least onenucleotide of the endogenous p-coumaroyl-CoA:monolignol transferase geneto generate at least one mutant plant cell with ap-coumaroyl-CoA:monolignol transferase gene knockdown or knockoutmutation; or

b) stably transforming one or plant cells with an expression cassettefor expression of an inhibitory nucleic acid adapted to hybridize to anendogenous p-coumaroyl-CoA:monolignol transferase nucleic transcript togenerate at least one transformed plant cell;

c) regenerating the mutant plant cell or the transformed plant cell intoat least one transgenic plant.

34B. The method of statement 33B, wherein the transgenic plant(s)comprises a recombinant feruloyl-CoA:monolignol transferase nucleic acidoperably linked to a promoter that expresses the feruloyl-CoA:monolignoltransferase protein in the transgenic plant.

35B. The method of statement 34B, wherein the transgenic plant hasincreased incorporation of monolignol ferulates into its lignin comparedto a control plant, wherein the control plant (a) does not have theknockout or knockdown mutation, (b) does not have the expressioncassette comprising an inhibitory nucleic acid, but (c) is stablytransformed with the recombinant feruloyl-CoA:monolignol transferasenucleic acid operably linked to a promoter that expresses theferuloyl-CoA:monolignol transferase protein.

36B. The method of any of statements 33B-35B, wherein the knockout orknockdown mutation, or the expression cassette comprising an inhibitorynucleic acid, increases incorporation of monolignol ferulates into thelignin of a plant by at least by 1%, or by at least 2%, or by at least3%, or by at least 5% relative to a control plant that (a) does not havethe knockout or knockdown mutation (b) does not have the expressioncassette comprising an inhibitory nucleic acid, but (c) is stablytransformed with the recombinant feruloyl-CoA:monolignol transferasenucleic acid operably linked to a promoter that expresses theferuloyl-CoA:monolignol transferase protein.

37B. The method of any of statements 33B-36B, wherein the endogenousp-coumaroyl-CoA:monolignol transferase gene can hybridize to a nucleicacid selected from the group consisting of SEQ ID NO:1 and 3; or theendogenous p-coumaroyl-CoA:monolignol transferase gene has at least 40%sequence identity, at least 45% sequence identity, at least 50% sequenceidentity, at least 55% sequence identity, at least 60% sequenceidentity, at least 65% sequence identity, at least 70% sequenceidentity, at least 75% sequence identity, at least 80% sequenceidentity, at least 85% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, or at least 97% sequenceidentity with a nucleic acid sequence selected from the group consistingof SEQ ID NO:1 and 3.

38B. The method of any of statements 33B-37B, wherein the mutatingnucleic acid has two flanking segments and a central segment,

wherein the central segment has a point mutation, a deletion, a missensemutation, or a nonsense mutation relative to a nucleic acid selectedfrom the group consisting of SEQ ID NO:1 and SEQ ID NO:3; and

wherein the two flanking segments can hybridize to different regions ofone of the nucleic acids selected from the group consisting of SEQ IDNO:1 and SEQ ID NO:3.

39B. The method of any of statements 33B-37B, wherein the inhibitorynucleic acid can selectively hybridize to a nucleic acid with a sequenceselected from the group consisting SEQ ID NO:1 and 3, and complementarysequences thereof

40B. The method of any of statements 33B-38B, wherein an inhibitorynucleic acid inhibits expression and/or translation of an endogenousp-coumaroyl-CoA:monolignol transferase mRNA expressed in at least onetransgenic plant.

41B. The method of any of statements 29B-40B, wherein the transgenicplant is fertile.

42B. The method of any of statements 29B-41B, further comprisingrecovering transgenic seeds from the transgenic plant.

43B. The method of any of statements 29B-42B, wherein the plant is amonocot.

44B. The method of any of statements 29B-33B, wherein the plant is agrass, maize or softwood plant.

45B. The method of any of statements 29B-44B, the plant is selected fromthe species consisting of Miscanthus giganteus, Panicum virgatum(switchgrass), Zea mays (corn), Oryza sativa (rice), Saccharum sp.(sugar cane), Triticum sp. (wheat), Avena sativa (oats), Pennisetumglaucum (pearl millet), Setaria italica (foxtail millet), Sorghum sp.(e.g., Sorghum bicolor), Bambuseae species (bamboo), Sorghastrum nutans(indiangrass), Tripsacum dactyloides (eastern gamagrass), Andropogongerardii (big bluestem), Schizachyrium scoparium (little bluestem),Bouteloua curtipendula (sideoats grama), Silphium terebinthinaceum(prairie rosinweed), Pseudoroegneria spicata (bluebunch wheatgrass)Sorghum bicolor (sorghum) and Bachypodium distachyon (purple falsebrome).

46B. The method of any of statements 29B-42B, wherein the plant is adicot, or hardwood.

47B. The method of any of statements 29B-46B, wherein the lignin in theplant comprises at least 1% monolignol ferulate, at least 2% monolignolferulate, at least 3% monolignol ferulate, at least 4% monolignolferulate, at least 5% monolignol ferulate, at least 10% monolignolferulate, at least 20% monolignol ferulate, or at least 25% monolignolferulate.

48B. The method of any of statements 29B-47B, wherein the lignin in theplant comprises about 1-30% monolignol ferulate, or about 2-30%monolignol ferulate.

49B. The method of any of statements 29B-48B, further comprisingbreeding a fertile transgenic plant to yield a progeny plant.

50B. The method of statement 49B, wherein the progeny plant compriseslignin with at least 1% monolignol ferulate, at least 2% monolignolferulate, at least 3% monolignol ferulate, at least 4% monolignolferulate, at least 5% monolignol ferulate, at least 10% monolignolferulate, at least 20% monolignol ferulate, or at least 25% monolignolferulate.

51B. The method of any of statements 29B-50B, further comprisingbreeding a fertile transgenic plant to yield a progeny plant that has anincrease in the percentage of monolignol ferulates in the lignin of theprogeny plant as a dominant trait while still maintaining functionalagronomic characteristics relative to the corresponding untransformedplant.

52B. The method of any of statements 29B-51B, further comprising stablytransforming the plant cell with at least one selectable marker gene.

53B. A fertile transgenic plant comprising a knockdown or knockoutmutation in an endogenous p-coumaroyl-CoA:monolignol transferase gene,and a recombinant feruloyl-CoA:monolignol transferase nucleic acidoperably linked to a promoter that expresses the feruloyl-CoA:monolignoltransferase protein.

54B. The fertile transgenic plant of statement 53B, wherein theknockdown or knockout mutation and the feruloyl-CoA:monolignoltransferase nucleic acid are transmitted through a complete normalsexual cycle of the transgenic plant to the next generation.

55B. A fertile transgenic plant stably transformed by the nucleic acidof any of statements 1B-11B, wherein the nucleic acid is operably linkedto a promoter functional in a host cell, wherein the nucleic acidexpresses an inhibitory nucleic acid and the nucleic acid is transmittedthrough a complete normal sexual cycle of the transgenic plant to thenext generation.

56B. The fertile transgenic plant of statement 55B, further comprising aferuloyl-CoA:monolignol transferase nucleic acid is transmitted througha complete normal sexual cycle of the transgenic plant to the nextgeneration.

57B. The fertile transgenic plant of any of statements 53B-56B, whereinthe plant is a monocot, grass, maize, gymnosperm or softwood.

58B. The fertile transgenic plant of any of statements 53B-57B, theplant is selected from the species consisting of Miscanthus giganteus,Panicum virgatum (switchgrass), Zea mays (corn), Oryza sativa (rice),Saccharum sp. (sugar cane), Triticum sp. (wheat), Avena sativa (oats),Pennisetum glaucum (pearl millet), Setaria italica (foxtail millet),Sorghum sp. (e.g., Sorghum bicolor), Bambuseae species (bamboo),Sorghastrum nutans (indiangrass), Tripsacum dactyloides (easterngamagrass), Andropogon gerardii (big bluestem), Schizachyrium scoparium(little bluestem), Bouteloua curtipendula (sideoats grama), Silphiumterebinthinaceum (prairie rosinweed), Pseudoroegneria spicata (bluebunchwheatgrass) Sorghum bicolor (sorghum) and Bachypodium distachyon (purplefalse brome).

59B. The fertile transgenic plant of any of statements 53B-56B, whereinthe plant is a dicot.

60B. The fertile transgenic plant of any of statements 53B-59B, whereinthe plant comprises lignin with at least 1% monolignol ferulate, atleast 2% monolignol ferulate, at least 3% monolignol ferulate, at least4% monolignol ferulate, at least 5% monolignol ferulate, at least 10%monolignol ferulate, at least 20% monolignol ferulate, or at least 25%monolignol ferulate.

61B. A lignin isolated from a transgenic plant comprising the isolatednucleic of any of statements 1B-12B, or the plant cell of statement 13B.

62B. A method of making a product from a transgenic plant comprising:

(a) providing or obtaining a transgenic plant that comprises an isolatednucleic acid encoding a feruloyl-CoA:monolignol transferase and (i) aknockdown or knockout mutation in an endogenousp-coumaroyl-CoA:monolignol transferase gene, or (ii) an expressioncassette for expression of an inhibitory nucleic acid adapted tohybridize to an endogenous p-coumaroyl-CoA:monolignol transferasenucleic transcript; and

(b) processing the transgenic plant's tissues under conditionssufficient to digest to the lignin to thereby generate the product fromthe transgenic plant;

wherein the transgenic plant's tissues comprise lignin having anincreased percent of monolignol ferulates relative to a correspondinguntransformed plant.

63B. The method of statement 62B, wherein the conditions sufficient todigest to the lignin comprise conditions sufficient to cleave esterbonds within monolignol ferulate-containing lignin.

64B. The method of statement 62B or 63B, wherein the conditionssufficient to digest to the lignin comprise mildly alkaline conditions.

65B. The method of any of statements 62B-64B, wherein the conditionssufficient to digest to the lignin comprise contacting the transgenicplant's tissues with ammonia for a time and a temperature sufficient tocleave ester bonds within monolignol ferulate-containing lignin.

66B. The method of any of statements 62B-65B, wherein the conditionssufficient to digest to the lignin would substantially not cleave etherand carbon-carbon bonds in lignin from a corresponding plant that doesnot contain the isolated nucleic acid encoding theferuloyl-CoA:monolignol transferase.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

Other embodiments are within the following claims.

We claim:
 1. A transgenic plant comprising an isolated nucleic acidmolecule, wherein the isolated nucleic acid molecule comprises anucleotide sequence encoding a polypeptide havingp-coumaroyl-CoA:monolignol transferase activity, wherein the polypeptidecomprises an amino acid sequence having at least 99% amino acid sequenceidentity to the amino acid sequence as set forth in SEQ ID NO:4, whereinthe nucleotide sequence is operably linked to a heterologous promoterfunctional or active in a plant cell, and wherein expression of thepolypeptide in the transgenic plant increases percent of monolignolcoumarates in the transgenic plant's lignin as compared to a controlplant of the same species lacking the isolated nucleic acid molecule andgrown under identical conditions.
 2. The transgenic plant of claim 1,wherein the transgenic plant does not have an increased percent ofmonolignol ferulates in the transgenic plant's lignin as compared to thecontrol plant.
 3. The transgenic plant of claim 1, wherein genome of thetransgenic plant is stably transformed with the isolated nucleic acidmolecule.
 4. The transgenic plant of claim 1, wherein the heterologouspromoter is functional or active during plant growth or development. 5.The transgenic plant of claim 1, wherein the heterologous promoter isfunctional or active in a woody tissue of a plant.
 6. A transgenic seedobtained from the transgenic plant of claim 1, wherein the transgenicseed comprises the isolated nucleic acid molecule.
 7. The transgenicplant of claim 1, wherein the polypeptide has the amino acid sequence asset forth in SEQ ID NO:4.
 8. A method for increasing a content ofmonolignol coumarates in lignin within a plant, comprising: (a) plantingthe transgenic seed of claim 6; and (b) cultivating a transgenic plantgerminated from the transgenic seed, wherein expression of thepolypeptide in the germinated transgenic plant increases the content ofmonolignol coumarates in the lignin within the germinated transgenicplant as compared to a control plant lacking the isolated nucleic acidmolecule and grown under identical growth conditions.
 9. A method ofobtaining a plant having increased content of monolignol coumarates inlignin within the plant, comprising the steps: (i) stably transformingplant cells with an isolated nucleic acid molecule, wherein the isolatednucleic acid molecule comprises a nucleotide sequence encoding apolypeptide having p-coumaroyl-CoA:monolignol transferase activity,wherein the polypeptide comprises an amino acid sequence having at least99% amino acid sequence identity to the amino acid sequence as set forthin SEQ ID NO:4, wherein the nucleotide sequence is operably linked to aheterologous promoter functional or active in a plant cell; and (ii)regenerating a transformed plant with the stably transformed plant cellsfrom step (i), wherein genome of the regenerated transgenic plant isstably transformed with the isolated nucleic acid molecule, and whereinthe regenerated transgenic plant has increased percent of monolignolcoumarates in the regenerated transgenic plant's lignin as compared to acontrol plant of the same species lacking the isolated nucleic acidmolecule and grown under identical conditions.
 10. The method of claim9, wherein the polypeptide has the amino acid sequence as set forth inSEQ ID NO:4.